SUMOylation Sites Research Articles

Sumoylation of the transcription factor Nrf2 (739.3)

The transcription factor Nrf2 induces transcription by binding to the antioxidant response element(s) in target gene promoters, enabling oxidatively stressed cells to mount a response to oxidative stress in order to restore redox homeostasis. To act as a transcription factor, Nrf2 must be separated from its inhibitory protein Keap1. Post‐translational modifications (PTMs) that mediate separation of Nrf2 from Keap1 have been extensively studied but PTMs that impact its biology after this separation are just beginning to emerge. Using in vitro sumoylation assay and other methods, we demonstrated that Nrf2 is a SUMO‐targeted protein [Malloy et al. (2013) JBC 288, 14569‐14583]. Here, we have used three SUMO site‐predicting algorithms (SeeSUMO, SUMOsp and SUMOplot) to locate putative SUMO‐binding sites in mouse Nrf2. Three of the fifteen putative sumoylation sites identified by these algorithms have high probability scores on all three prediction algorithms. Using site‐directed mutagenesis, Co‐IP, and in vitro sumoylation assays, we investigated which of these sites is/are authentic for SUMO conjugation. We also assessed whether sumoylating Nrf2 impacts its transcriptional activity.Grant Funding Source: Supported by NIH grants SC1CA143985, 5T32 GM07628‐34 and CTSA award No.UL1TR000445 from the NCATS.

The conserved sumoylation consensus site in TRIM5α modulates its immune activation functions

TRIM5α is a type I interferon-stimulated anti-retroviral restriction factor expressed in most primates and homologous proteins are expressed in other mammals. Through its C-terminal PRYSPRY (B30.2) domain, TRIM5α binds to incoming and intact post-fusion retroviral cores in the cytoplasm. Following this direct interaction, the retroviral capsid core is destabilized and progression of the virus life cycle is interrupted. Specific recognition of its viral target by TRIM5α also triggers the induction of an antiviral state involving the activation of transcription factors NF-κB- and AP-1. In addition to PRYSPRY, several other TRIM5α domains are important for anti-retroviral function, including a RING zinc-binding motif. This domain has “E3” ubiquitin ligase activity and is involved in both the direct inhibition of incoming retroviruses and innate immune activation. A highly conserved sumoylation consensus site is present between the RING motif and the N-terminal extremity of TRIM5α. No clear role in restriction has been mapped to this sumoylation site, and no sumoylated forms of TRIM5α have been observed. Here we confirm that mutating the putatively sumoylated lysine (K10) of the Rhesus macaque TRIM5α (TRIM5αRh) to an arginine has only a small effect on restriction. However, we show that the mutation significantly decreases the TRIM5α-induced generation of free K63-linked ubiquitin chains, an intermediate in the activation of innate immunity pathways. Accordingly, K10R decreases TRIM5α-mediated activation of both NF-κB and AP-1. Concomitantly, we find that K10R causes a large increase in the levels of ubiquitylated TRIM5α. Finally, treatment with the nuclear export inhibitor leptomycin B shows that K10R enhances the nuclear localization of TRIM5αRh, while at the same time reducing its level of association with nuclear SUMO bodies. In conclusion, the TRIM5α sumoylation site appears to modulate the E3 ubiquitin ligase activities of the adjacent RING domain, promoting K63-linked ubiquitin chains at the expense of auto-ubiquitylation which is probably K48-linked. Consistently, we find this sumoylation site to be important for innate immune activation by TRIM5α. In addition, lysine 10 regulates TRIM5α nuclear shuttling and nuclear localization, which may also be related to its role in innate immunity activation.

Isolation, molecular cloning and characterization of a cold-responsive gene, AmDUF1517, from Ammopiptanthus mongolicus

Temperature is one of important factors that influence plant growth and development. Using cDNA-amplified fragment length polymorphism approach, we previously screened 344 temperature-related transcript-derived fragments (TDFs) from Ammopiptanthus mongolicus. In this study, we confirmed that 15 of these TDFs were upregulated in response to low- or high-temperature by using semi-quantitative RT-PCR. Based on the rapid amplification of cDNA ends, PCR and genome walking approaches, full-length cDNA and promoter sequence of AmDUF1517 was cloned and identified. The 906 bp open reading frame of the AmDUF1517 gene encoded for a protein of 301 amino acids residues. The corresponding genomic DNA sequence contains two exons and one intron. Bioinformatic analysis showed that a predicted cleavage site for chloroplast transit peptide, a DUF1517 domain, two transmembrane domains and two putative sumoylation sites were conserved between AmDUF1517 and its homolog from Arabidopsis thaliana (AtDUF1517). We further showed that GFP-tagged AmDUF1517 was indeed targeted to the chloroplast in Arabidopsis protoplast. The transcript levels of AmDUF1517 were increased specifically in leaves in response to cold stress. In addition, treatment of ethylene, salicylic acid, gibberellic acid or NaCl induced the transcription of AmDUF1517. Mutation of DUF1517 in Arabidopsis exhibited enhanced sensitivity to cold stress, which was coupled with increased electrolyte leakage, malondialdehyde content and decreased contents of soluble sugar, proline. Interestingly, heterologous expression of AmDUF1517 in Arabidopsisatduf1517 mutants significantly rescued their cold-sensitive phenotypes. Altogether, our data suggest the potential roles of both AmDUF1517 and AtDUF1517 in the regulation of cold stress tolerance.

Abstract P2-06-02: Pparg deacetylation by SIRT1 determines breast tumor lipid synthesis and growth

Abstract Peroxisome proliferator-activated receptorg (Pparγ) is a member of the nuclear receptor (NR) superfamily, which regulates diverse biological functions including lipogenesis and differentiation, anti-inflammation, insulin sensitivity, cellular proliferation, and autophagy. Independent lines of evidence support a role for Pparγ as either a collaborative oncogene or as a tumor suppressor. Heterozygous mutations of Pparγ have been detected in 4/55 patients with colon cancer and a chromosomal translocation between PAX8 and Pparγ in follicular thyroid cancer appeared to serve as a dominant inhibitor of endogenous Pparγ expression. Pparγ agonists reduced tumorigenesis in several in vivo models. In contrast, several studies suggest Pparγ may enhance tumor growth. Pparγ ligands increased polyp numbers in the Apc mouse model of familial adenomatosis. Pparγ and its ligands inhibit breast tumor growth; however, constitutively active Pparγ collaborated in mammary oncogenesis with polyoma middle T antigen or oncogenic ErbB2. Pparγ activation involves post-translational modifications including phosphorylation and sumoylation upon growth factor or ligand stimulus. Mutation of the Pparγ1 sumoylation site at K77 and K365 demonstrated that K77 may either reduce Pparγ-dependent gene induction and enhance repression or reduce repression, depending upon the synthetic reporter gene used. Lysine residues of nuclear receptors also serve as substrates for acetylation and Pparγ binds co-activators and co-repressors with intrinsic or associated histone acetylase or deacetylase activity including NCoR, SMRT, SIRT1, and p300. Initially characterized for the ERα, AR and, subsequently, the orphan nuclear receptor steroidogenic factor 1 (SF-1), acetylation occurs at a conserved lysine motif shared amongst evolutionarily related nuclear receptors. Several nuclear receptors and co-integrators involved in lipid metabolism are regulated by acetylation including p300, PGC1α, FXR, LXR and RAR. Both TSA- and NAD-sensitive HDACs (e.g. SIRT1) regulate Pparγ function and SIRT1 inhibits Pparγ-dependent adipocyte differentiation. Whether Pparγ is acetylated in cancer cells and how Pparγ exerts it's crucial, though controversial, function in tumorigenesis have not been established. Pparγ induces gene transcription through binding specific NR half-sites and through non-canonical binding sequences (such as CREB/AP-1 sites). Transcriptional repression involves Pparγ sumoylation at lysine 77 (K77). Herein, Pparγ was shown to be acetylated at nine distinct lysine residues. SIRT1 bound and deacetylated Pparγ at K154/155. ChIP-Seq analysis for genome-wide DNA binding demonstrated the acetylation site was required for binding NR half-sites, but was not required for non-canonical site binding. Breast tumor growth, de novo lipid synthesis, induction of autophagy and evasion of apoptosis was promoted by K154/155 and inhibited by K77 in vivo. Pparγ acetylation induced a gene signature that was increased in breast cancer, associated with a reduction in SIRT1 abundance and poor outcome. The Pparγ acetylation site determines binding to autophagy and apoptosis signaling to regulate breast tumor lipid metabolism and growth. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-02.

Stress-induced Phosphorylation of Thr486 in c-Myb by p38 Mitogen-activated Protein Kinases Attenuates Conjugation of SUMO-2/3

c-Myb plays an essential role in regulation of properly balanced hematopoiesis through transcriptional regulation of genes directly controlling cellular processes such as proliferation, differentiation, and apoptosis. The transcriptional activity and protein levels of c-Myb are strictly controlled through post-translational modifications such as phosphorylation, acetylation, ubiquitination, and SUMOylation. Conjugation of small ubiquitin-like modifier (SUMO) proteins has been shown to suppress the transcriptional activity of c-Myb. SUMO-1 modifies c-Myb under physiological conditions, whereas SUMO-2/3 conjugation was reported in cells under stress. Because stress also activates several cellular protein kinases, we investigated whether phosphorylation of c-Myb changes in stressed cells and whether a mutual interplay exists between phosphorylation and SUMOylation of c-Myb. Here we show that several types of environmental stress induce a rapid change in c-Myb phosphorylation. Interestingly, the phosphorylation of Thr(486), located in close proximity to SUMOylation site Lys(499) of c-Myb, is detected preferentially in nonSUMOylated protein and has a negative effect on stress-induced SUMOylation of c-Myb. Stress-activated p38 MAPKs phosphorylate Thr(486) in c-Myb, attenuate its SUMOylation, and increase its proteolytic turnover. Stressed cells expressing a phosphorylation-deficient T486A mutant demonstrate decreased expression of c-Myb target genes Bcl-2 and Bcl-xL and accelerated apoptosis because of increased SUMOylation of the mutant protein. These results suggest that phosphorylation-dependent modulation of c-Myb SUMOylation may be important for proper response of cells to stress. In summary, we have identified a novel regulatory interplay between phosphorylation and SUMOylation of c-Myb that regulates its activity in stressed cells.

Notch Alters Sumoylation To Govern GFI1 Protein Stability and Support Its Transcriptional Repression Function

The zinc-finger (ZF) transcriptional repressors, Growth factor independence (GFI)-1 and -1B are master regulators of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. GFI family members share near invariant SNAG and ZF domains at their N- and C-termini, respectively. Their SNAG domains recruit Lysine Specific Demethylase (LSD)-1, a dominant effector of transcriptional repression by GFI proteins, while ZFs 3-5 of both GFI1 and GFI1B recognize a common consensus element in target promoters. A non-homologous linker separates the SNAG and ZF domains. Despite near identical DNA binding and co-factor recruitment characteristics, GFI family proteins direct distinct hematopoietic outcomes. Granulopoiesis requires Gfi1, and Gfi1 mutations cause severe congenital neutropenia (SCN) type II. Gfi1 is also necessary for T-lymphopoiesis and maintenance of the malignant phenotype in lymphoid leukemias and lymphomas. In contrast, Gfi1b specifies erythroid and megakaryocytic fates and is often abundantly expressed in myeloid leukemias. These distinct outcomes cannot be explained by patterns of expression alone, as Gfi1 and Gfi1B transcripts partially overlap during hematopoiesis. These findings suggest a need to distinguish between GFI family proteins as they integrate developmental signals to coordinate alternate hematopoietic fates. How this occurs is not understood. We hypothesized that functional differences between GFI1 and GFI1B may arise from interactions and post-translational modifications occurring uniquely within their linker regions. We show the GFI1 linker contains a type-I SUMOylation consensus motif centered on K239 that is absolutely conserved among mammalian GFI1 proteins but absent from GFI1B. K239 is required for robust GFI1 SUMOylation, as arginine substitution (K239R) significantly impairs formation of high molecular weight, SUMO-conjugated GFI1 derivatives. Coincidentally, K239R substitution also significantly impairs LSD1 recruitment by the GFI1 SNAG domain. The SUMOylation site in the GFI1 linker is contained within the binding site for PIAS3, an E3 SUMO ligase. PIAS3 co-expression antagonizes transcriptional repression by GFI1, but not GFI1B. The GFI1-K239R derivative represses transcription comparable to wild type GFI1, but resists the inhibitory effects of PIAS3. Moreover, GFI1-K239R preferentially co-purifies with chromatin in fractionated cells, displaying a prolonged half-life relative to wild type GFI1. These data suggest that GFI1 SUMOylation occurs on chromatin, facilitates LSD1 recruitment and serves as a prelude to GFI1 degradation. We surmised that the Notch1 intracellular domain (N1-ICD), which requires GFI1 for both normal and malignant T-lymphopoiesis, might alter GFI1 SUMOylation to reinforce its functions as a transcriptional regulator. We show that N1-ICD interacts directly with the GFI1 linker via the same motif that binds PIAS3. N1-ICD and PIAS3 compete for GFI1 binding and display an oppositional relationship in GFI1-mediated transcriptional repression. N1-ICD reinforces GFI1 as a transcriptional repressor, blocks GFI1 conjugation by ubiquitin family proteins and prevents GFI1 degradation. Our findings suggest a post-transcriptional mechanism by which Notch signaling endorses GFI1 as a transcriptional regulator and intimate GFI1 binding partners like LSD1 as candidate biological response modifiers in Notch-driven malignancies. Disclosures:No relevant conflicts of interest to declare.

FLC-mediated flowering repression is positively regulated by sumoylation

Flowering locus C (FLC), a floral repressor, is a critical factor for the transition from the vegetative to the reproductive phase. Here, the mechanisms regulating the activity and stability of the FLC protein were investigated. Bimolecular fluorescence complementation and in vitro pull-down analyses showed that FLC interacts with the E3 small ubiquitin-like modifier (SUMO) ligase AtSIZ1, suggesting that AtSIZ1 is an E3 SUMO ligase for FLC. In vitro sumoylation assays showed that FLC is modified by SUMO in the presence of SUMO-activating enzyme E1 and conjugating enzyme E2, but its sumoylation is inhibited by AtSIZ1. In transgenic plants, inducible AtSIZ1 overexpression led to an increase in the concentration of FLC and delayed the post-translational decay of FLC, indicating that AtSIZ1 stabilizes FLC through direct binding. Also, the flowering time in mutant FLC (K154R, a mutation of the sumoylation site)-overexpressing plants was comparable with that in the wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. The data indicate that the sumoylation of FLC is critical for its role in the control of flowering time and that AtSIZ1 positively regulates FLC-mediated floral suppression.

Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.

Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination

The cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. Here, we show that stimulation of cultured mouse cerebellar neurons by a function-triggering L1 antibody leads to cathepsin E-mediated generation of a sumoylated 30kDa L1 fragment (L1-30) and to import of L1-30 into the nucleus. Mutation of the sumoylation site at K1172 or the cathepsin E cleavage site at E1167 abolishes generation of L1-30, while mutation of the nuclear localization signal at K1147 prevents nuclear import of L1-30. Moreover, the aspartyl protease inhibitor pepstatin impairs the generation of L1-30 and inhibits L1-induced migration of cerebellar neurons and Schwann cells as well as L1-dependent in vitro myelination on axons of dorsal root ganglion neurons by Schwann cells. L1-stimulated migration of HEK293 cells expressing L1 with mutated cathepsin E cleavage site is diminished in comparison to migration of cells expressing non-mutated L1. In addition, L1-stimulated migration of HEK293 cells expressing non-mutated L1 is also abolished upon knock-down of cathepsin E expression and enhanced by over-expression of cathepsin E. The findings of the present study indicate that generation and nuclear import of L1-30 regulate neuronal and Schwann cell migration as well as myelination. Cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. L1 stimulation triggers sumoylation and cleavage of L1, thus generating the L1-70 fragment (1) which is cleaved by cathepsin E (2) yielding the L1-30 fragment that is imported to the nucleus (3), may bind to DNA and/or nuclear proteins (4), to regulate diverse cellular functions.

The function of EHD2 in endocytosis and defense signaling is affected by SUMO

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX.

TAB2, an important upstream adaptor of interleukin-1 signaling pathway, is subject to SUMOylation

SUMOylation has been considered as an important mechanism to regulate multiple cellular processes, including inflammation. TAB2 (TAK1-binding protein 2) is an upstream adaptor protein in the IL-1 signaling pathway. Covalent modifications of TAB2 have not been well studied. In this study, we demonstrated that TAB2 could be modified by SUMO. Using Ubc9 (SUMO-conjugating enzyme) fusion and mutation analysis, we identified evolutionarily conserved lysine 329 as the major SUMOylation site of TAB2. PIAS3, a SUMO E3 ligase, preferentially interacted with and promoted its SUMOylation. Interestingly, block of SUMOylation by mutation of lysine 329 enhanced the activity of TAB2, as reflected by AP-1 luciferase reporter assays. Taken together, these results suggest that SUMOylation may serve as a novel mechanism for the regulation of TAB2.

Zebrafish brain proteomics reveals central proteins involved in neurodegeneration

Understanding the complex biology of the brain requires analyzing its structural and functional complexity at the protein level. The large-scale analysis of the brain proteome, coupled with characterization of central brain proteins, provides insight into fundamental brain processes and processes linked to neurodegenerative diseases. Here we provide a map of the zebrafish brain proteome by using two-dimensional gel electrophoresis (2DE), followed by the identification of 95 brain proteins using mass spectrometry (LC-ESI MS/MS). Our data show extensive phosphorylation of brain proteins but less prominent glycosylation. Furthermore, ~51% of the identified proteins are predicted to have one or more ubiquitination sites whereas ~90% are predicted to have one or more SUMOylation sites. Our findings provide a valuable proteome map of the zebrafish brain and associated posttranslational modifications demonstrating that zebrafish proteomic approaches can aid in our understanding of proteins central to important neuronal processes and those associated with neurodegenerative disorders.

Targeted Identification of SUMOylation Sites in Human Proteins Using Affinity Enrichment and Paralog-specific Reporter Ions

Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications.

RSUME Enhances Glucocorticoid Receptor SUMOylation and Transcriptional Activity

Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant.

Development of a High-Throughput Screen to Detect Inhibitors of TRPS1 Sumoylation

Small ubiquitin-like modifier (SUMO) belongs to the family of ubiquitin-like proteins (Ubls) that can be reversibly conjugated to target-specific lysines on substrate proteins. Although covalently sumoylated products are readily detectible in gel-based assays, there has been little progress toward the development of robust quantitative sumoylation assay formats for the evaluation of large compound libraries. In an effort to identify inhibitors of ubiquitin carrier protein 9 (Ubc9)-dependent sumoylation, a high-throughput fluorescence polarization assay was developed, which allows detection of Lys-1201 sumoylation, corresponding to the major site of functional sumoylation within the transcriptional repressor trichorhino-phalangeal syndrome type I protein (TRPS1). A minimal hexapeptide substrate peptide, TMR-VVK₁₂₀₁TEK, was used in this assay format to afford high-throughput screening of the GlaxoSmithKline diversity compound collection. A total of 728 hits were confirmed but no specific noncovalent inhibitors of Ubc9 dependent trans-sumoylation were found. However, several diaminopyrimidine compounds were identified as inhibitors in the assay with IC₅₀ values of 12.5 μM. These were further characterized to be competent substrates which were subject to sumoylation by SUMO-Ubc9 and which were competitive with the sumoylation of the TRPS1 peptide substrates.

Covalent Small Ubiquitin-like Modifier (SUMO) Modification of Maf1 Protein Controls RNA Polymerase III-dependent Transcription Repression

RNA polymerase (pol) III transcribes genes that determine biosynthetic capacity. Induction of these genes is required for oncogenic transformation. The transcriptional repressor, Maf1, plays a central role in the repression of these and other genes that promote oncogenesis. Our studies identify an important new role for SUMOylation in repressing RNA pol III-dependent transcription. We show that a key mechanism by which this occurs is through small ubiquitin-like modifier (SUMO) modification of Maf1 by both SUMO1 and SUMO2. Mutation of each lysine residue revealed that Lys-35 is the major SUMOylation site on Maf1 and that the deSUMOylase, SENP1, is responsible for controlling Maf1K35 SUMOylation. SUMOylation of Maf1 is unaffected by rapamycin inhibition of mammalian target of rapamycin (mTOR) and mTOR-dependent Maf1 phosphorylation. By preventing SUMOylation at Lys-35, Maf1 is impaired in its ability to both repress transcription and suppress colony growth. Although SUMOylation does not alter Maf1 subcellular localization, Maf1K35R is defective in its ability to associate with RNA pol III. This impairs Maf1 recruitment to tRNA gene promoters and its ability to facilitate the dissociation of RNA pol III from these promoters. These studies identify a novel role for SUMOylation in controlling Maf1 and RNA pol III-mediated transcription. Given the emerging roles of SENP1, Maf1, and RNA pol III transcription in oncogenesis, our studies support the idea that deSUMOylation of Maf1 and induction of its gene targets play a critical role in cancer development.

Multidomain sumoylation of the ecdysone receptor (EcR) from Drosophila melanogaster

The 20-hydroxyecdysone receptor (EcR) is a transcription factor belonging to the nuclear receptor superfamily. Together with the ultraspiracle nuclear receptor (Usp) it coordinates critical biological processes in insects such as development and reproduction. EcR and its ligands are used in commercially available ecdysone-inducible expression systems and are considered to be artificial gene switches with potential therapeutic applications. However, the regulation of EcR action is still unclear, especially in mammals and as far as posttranslational modifications are concerned. Up until now, there has been no study on EcR sumoylation. Using bioinformatic predictors, a Ubc9 fusion-directed sumoylation system and mutagenesis experiments, we present EcR as a new target of SUMO1 and SUMO3 modification. Our research revealed that EcR undergoes isoform-specific multisumoylation. The pattern of modification remains unchanged in the presence of the ligand and the dimerization partner. The SUMO acceptor sites are located in the DNA-binding domain and the ligand-binding domain that both exhibit structural plasticity. We also demonstrated the existence of a sumoylation site in the F region and EcRA-A/B region, both revealing characteristics of intrinsically disordered regions. The consequences of modification and the resulting impact on conformation and function may be especially crucial for the disordered sequences in these two areas. The isoform-specificity of sumoylation may explain the differences in the transcriptional activity of EcR isoforms.

SUMOylation of Mouse p53b by SUMO-1 Promotes Its Pro-Apoptotic Function in Ovarian Granulosa Cells

Follicular atresia is a process of spontaneous degradation of follicles, hindering growth and development in the mammalian ovary. Previous studies showed that follicular atresia was caused by apoptosis of granulosa cells, for which a number of apoptosis-related genes have already been identified. The roles of p53 in apoptosis of mouse granulosa cells and its post-translational modification are still unclear. The main objective of this study was to explore the roles of p53 in mouse granulosa cells. We found that mouse p53b, but not p53a, could be SUMOylated by SUMO-1 at lysine 375, which was essential for the protein stability of p53b in a dose-dependent manner. Immunofluorescent staining showed that wild p53b was located in the nucleus of granulosa cells, while its mutation of SUMOylated site (K375R) was localized in both nucleus and cytoplasm, implying that SUMOylation was necessary for the nuclear localization of p53b in granulosa cells. Overexpression of wild-type p53b, but not the mutation of SUMOylation site (K375R), significantly induced the expression of apoptosis-related gene, Bax, and increased the level of apoptosis in granulosa cells. This suggested that SUMO-1 modification of p53b was essential for inducing apoptosis in granulosa cells. Our results provide strong evidences that modification of p53b by SUMO-1 at lysine 375 was necessary for its activity to induce apoptosis in mouse granulosa cells, and it was involved in the regulation of p53b protein stability and nuclear localization. This implies that modification of p53b by SUMO-1 might regulate follicular atresia by inducing the apoptosis of ovarian granulosa cells in mice.

Sumoylation of Krüppel-like Factor 4 Inhibits Pluripotency Induction but Promotes Adipocyte Differentiation

Ectopic expression of transcription factors has been shown to reprogram somatic cells into induced pluripotent stem (iPS) cells. It remains largely unexplored how this process is regulated by post-translational modifications. Several reprogramming factors possess conserved sumoylation sites, so we investigated whether and how this modification regulates reprogramming of fibroblasts into iPS cells. Substitution of the sole sumoylation site of the Krüppel-like factor (KLF4), a well known reprogramming factor, promoted iPS cell formation. In comparison, much smaller effects on reprogramming were observed for sumoylation-deficient mutants of SOX2 and OCT4, two other classical reprogramming factors. We also analyzed KLF2, a KLF4 homolog and a member of the KLF family of transcription factors with a known role in reprogramming. KLF2 was sumoylated at two conserved neighboring motifs, but substitution of the key lysine residues only stimulated reprogramming slightly. KLF5 is another KLF member with an established link to embryonic stem cell pluripotency. Interestingly, although it was much more efficiently sumoylated than either KLF2 or KLF4, KLF5 was inactive in reprogramming, and its sumoylation was not responsible for this deficiency. Furthermore, sumoylation of KLF4 but not KLF2 or KLF5 stimulated adipocyte differentiation. These results thus demonstrate the importance KLF4 sumoylation in regulating pluripotency and adipocyte differentiation.

Regulation of IL-4-induced STAT6 activation by SUMOylation (P6307)

Abstract As a key signaling molecule in the cytokine-mediated transcriptional activation STATs are shown to undergo PTMs including phosphorylation, acetylation, methylation, ubiquitination and SUMOylation. The SUMO-modification of STAT1 has been implicated in the regulation of protein stability, nuclear translocation, and transcriptional activation, probably through the reduction of the formation and life-span of the active dimer. As a part of regulation of IL-4 signaling mechanism, we have investigated the role of SUMOylation on STAT6 activation. We have identified potential SUMOylation sites on STAT6 by SUMOplot prediction program analysis and observed the IL-4-induced SUMOlyation of STAT6 in HeLa cells. The introduction of mutation at a potential SUMO site K636 and transfection of shRNA of SUMO-conjugating enzyme UBC9 resulted in the inhibition of tyrosine phosphorylation at Y641 induced by IL-4. The results indicate that endogenous SUMOylation reaction of STAT6 is critical for IL-4-induced STAT6 phosphorylation. Conversely, the forced expression of SUMO-1 and STAT6 caused cytosolic relocalization of both STAT6 and phospho-STAT6 induced by IL-4 as demonstrated by Western blot and confocal analysis. Our data suggest that SUMO-modification of STAT6 may play dual roles in the regulation of phosphorylation and nuclear translocation of STAT6 during IL-4 signal transduction.