Small Ubiquitin-related Modifier Conjugation Research Articles

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

Small ubiquitin-related modifier (SUMO) pathway-mediated enhancement of human cytomegalovirus replication correlates with a recruitment of SUMO-1/3 proteins to viral replication compartments

Recent studies have suggested that the small ubiquitin-related modifier (SUMO) conjugation pathway may play an important role in intrinsic antiviral resistance and thus for repression of herpesviral infections. In particular, it was shown that the herpes simplex virus type-1 regulatory protein ICP0 acts as a SUMO-targeted ubiquitin ligase (STUbL), inducing the widespread degradation of SUMO-conjugated proteins during infection. As the IE1 protein of human cytomegalovirus (HCMV) is known to mediate a de-SUMOylation of PML, we investigated whether HCMV uses a similar mechanism to counteract intrinsic antiviral resistance. We generated primary human fibroblasts stably expressing FLAG-SUMO-1 or FLAG-SUMO-3 and analysed the SUMOylation pattern after HCMV infection or isolated IE1 expression. However, Western blot experiments did not reveal a global loss of SUMO conjugates, either in HCMV-infected or in IE1-expressing cells, arguing against a function of IE1 as an STUbL. Interestingly, we observed that FLAG-SUMO-1 and FLAG-SUMO-3, subsequent to IE1-mediated promyelocytic leukemia protein (PML) de-SUMOylation and the consequent disruption of PML nuclear bodies, were recruited into viral replication compartments. This raised the question of whether FLAG-SUMO-1/3 might promote HCMV replication. Intriguingly, overexpression of FLAG-SUMO-1/3 enhanced accumulation of viral DNA, which correlated with an increase in viral replication and in virus particle release. Together, these data indicate that HCMV, in contrast to other herpesviruses, has evolved subtle mechanisms enabling it to utilize the SUMO conjugation pathway for its own benefit, resulting in an overall positive effect of SUMO conjugation for HCMV replication.

SUMOylation in Control of Accurate Chromosome Segregation during Mitosis

Posttranslational protein modification by small ubiquitin-related modifier (SUMO) has emerged as an important regulatory mechanism for chromosome segregation during mitosis. This review focuses on how SUMOylation regulates the centromere and kinetochore activities to achieve accurate chromosome segregation during mitosis. Kinetochores are assembled on the specialized chromatin domains called centromeres and serve as the sites for attaching spindle microtubule to segregate sister chromatids to daughter cells. Many proteins associated with mitotic centromeres and kinetochores have been recently found to be modified by SUMO. Although we are still at the early stage of elucidating how SUMOylation controls chromosome segregation during mitosis, a substantial progress has been achieved over the past decade. Furthermore, a major theme that has emerged from the recent studies of SUMOylation in mitosis is that both SUMO conjugation and deconjugation are critical for kinetochore assembly and disassembly. Lastly, we propose a model that SUMOylation coordinates multiple centromere and kinetochore activities to ensure accurate chromosome segregation.

SUMOylation in Drosophila Development.

Small ubiquitin-related modifier (SUMO), an ~90 amino acid ubiquitin-like protein, is highly conserved throughout the eukaryotic domain. Like ubiquitin, SUMO is covalently attached to lysine side chains in a large number of target proteins. In contrast to ubiquitin, SUMO does not have a direct role in targeting proteins for proteasomal degradation. However, like ubiquitin, SUMO does modulate protein function in a variety of other ways. This includes effects on protein conformation, subcellular localization, and protein–protein interactions. Significant insight into the in vivo role of SUMOylation has been provided by studies in Drosophila that combine genetic manipulation, proteomic, and biochemical analysis. Such studies have revealed that the SUMO conjugation pathway regulates a wide variety of critical cellular and developmental processes, including chromatin/chromosome function, eggshell patterning, embryonic pattern formation, metamorphosis, larval and pupal development, neurogenesis, development of the innate immune system, and apoptosis. This review discusses our current understanding of the diverse roles for SUMO in Drosophila development.

The Role of the Small Ubiquitin-Related Modifier (SUMO) Pathway in Prostate Cancer

SUMO (small ubiquitin-related modifier) conjugation is a reversible three-step process of protein post-translational modifications mediating protein-protein interactions, subcellular compartmentalization and regulation of transcriptional events. Among divergent transcription factors regulated by SUMOylation and deSUMOylation, the androgen receptor (AR) is of exceptional significance, given its established role in prostate carcinogenesis. The enzymes of the SUMO pathway can have diverse effects on AR transcriptional activity, either via direct modification of the AR or through modification of AR co-regulators. Accumulating in vitro and in vivo evidence implicates the SUMO pathway in AR-dependent signaling. Prostate cancer cell proliferation and hypoxia-induced angiogenesis are also regulated by the SUMO pathway, through an AR-independent mechanism. Thus, an important role has been revealed for members of the SUMO pathway in prostate cancer (PCa) development and progression, offering new therapeutic targets.

A stress inducible SUMO conjugating enzyme gene (SaSce9) from a grass halophyte Spartina alterniflora enhances salinity and drought stress tolerance in Arabidopsis

BackgroundSUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene ‘SaSce9’ from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance.ResultsSaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of ~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22.ConclusionsThese results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement.

Small Ubiquitin-related Modifier (SUMO)-1 Promotes Glycolysis in Hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress.

The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition

Proteins of the small ubiquitin-related modifier (SUMO) family are conjugated to proteins to regulate such cellular processes as nuclear transport, transcription, chromosome segregation and DNA repair. Recently, numerous insights into regulatory mechanisms of the SUMO modification pathway have emerged. Although SUMO-conjugating enzymes can discriminate between SUMO targets, many substrates possess characteristics that facilitate their modification. Other post-translational modifications also regulate SUMO conjugation, suggesting that SUMO signalling is integrated with other signal transduction pathways. A better understanding of SUMO regulatory mechanisms will lead to improved approaches for analysing the function of SUMO and substrate conjugation in distinct cellular pathways.

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

Sumoylation and abscisic acid signaling

The conjugation of small ubiquitin-related modifier (SUMO) to substrates (sumoylation) is one of posttranslational modification systems in eukaryotes. Sumoylation plays an important role in the regulation of environmental stress response, biotic stress response, and flowering control in plants. Covalent SUMO conjugation requires an E1/E2/E3 enzyme, and SUMO E3 ligase SIZ1 is essential for these regulations. This addendum summarizes our recent study in which it has been established that in Arabidopsis, SUMO E3 ligase SIZ1 negatively controls abscisic acid (ABA) signaling through the sumoylation of ABI5. The conjugation of SUMO to ABI5 represses its activity and also prevents ABI5 from undergoing degradation.

Specific Domain Structures Control Abscisic Acid-, Salicylic Acid-, and Stress-Mediated SIZ1 Phenotypes

SIZ1 (for yeast SAP and MIZ1) encodes the sole ortholog of mammalian PIAS (for protein inhibitor of activated STAT) and yeast SIZ SUMO (for small ubiquitin-related modifier) E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (for scaffold attachment factor A/B/acinus/PIAS domain), PINIT (for proline-isoleucine-asparagine-isoleucine-threonine), SP-RING (for SIZ/PIAS-RING), and SXS (for serine-X-serine, where X is any amino acid) motifs. SIZ1 contains, in addition, a PHD (for plant homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. Salicylic acid (SA) accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity, and abscisic acid inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of abscisic acid. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment.

Sumoylation of ABI5 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid signaling

SUMO (small ubiquitin-related modifier) conjugation (i.e., sumoylation) to protein substrates is a reversible posttranslational modification that regulates signaling by modulating transcription factor activity. This paper presents evidence that the SUMO E3 ligase SIZ1 negatively regulates abscisic acid (ABA) signaling, which is dependent on the bZIP transcripton factor ABI5. Loss-of-function T-DNA insertion siz1-2 and siz1-3 mutations caused ABA hypersensitivity for seed germination arrest and seedling primary root growth inhibition. Furthermore, expression of genes that are ABA-responsive through ABI5-dependent signaling (e.g., RD29A, Rd29B, AtEm6, RAB18, ADH1) was hyperinduced by the hormone in siz1 seedlings. abi5-4 suppressed ABA hypersensitivity caused by siz1 (siz1-2 abi5-4), demonstrating an epistatic genetic interaction between SIZ1 and ABI5. A K391R substitution in ABI5 [ABI5(K391R)] blocked SIZ1-mediated sumoylation of the transcription factor in vitro and in Arabidopsis protoplasts, indicating that ABI5 is sumoylated through SIZ1 and that K391 is the principal site for SUMO conjugation. In abi5-4 plants, ABI5(K391R) expression caused greater ABA hypersensitivity (gene expression, seed germination arrest and primary root growth inhibition) compared with ABI5 expression. Together, these results establish that SIZ1-dependent sumoylation of ABI5 attenuates ABA signaling. The double mutant siz1-2 afp-1 exhibited even greater ABA sensitivity than the single mutant siz1, suggesting that SIZ1 represses ABI5 signaling function independent of AFP1.

PIASy inhibits LRH-1-dependent CYP11A1 expression by competing for SRC-1 binding

The orphan nuclear receptor LRH-1 (liver receptor homologue-1; NR5A2) plays a critical role in development, bile acid synthesis and cholesterol metabolism. LRH-1 is also expressed in the ovary where it is implicated in the regulation of steroidogenic genes for steroid hormone synthesis. In the present study, we investigated the molecular mechanisms of the transcriptional regulation of CYP11A1 by LRH-1 and found that LRH-1-mediated transactivation was markedly repressed by PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) y], the shortest member of the PIAS family. The suppression of LRH-1 activity requires the N-terminal repression domain. Although PIAS proteins also function as E3 SUMO (small ubiquitin-related modifier) ligases and enhance SUMO conjugation, PIASy-mediated repression was independent of LRH-1 SUMOylation status. In addition, histone deacetylase activity was not involved in the inhibition of LRH-1 by PIASy. Immunoprecipitation and mammalian two-hybrid analyses indicated that PIASy interacted with LRH-1 through the C-terminal region, including the AF-2 (activation function-2) motif, which was also involved in the interaction between LRH-1 and the co-activator SRC-1 (steroid receptor co-activator-1). PIASy inhibited the binding of SRC-1 to LRH-1, although overexpression of SRC-1 partially overcame the PIASy inhibition of LRH-1 induction of the CYP11A1 promoter. The results of the present study suggest that competition with co-activators may be an important mechanism underlying the PIASy repression of LRH-1-mediated transactivation.

Liver receptor homolog‐1 localization in the nuclear body is regulated by sumoylation and cAMP signaling in rat granulosa cells

Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the nuclear receptor superfamily, mainly expressed in endoderm-derived tissues and in the ovary. In ovarian granulosa and luteal cells, LRH-1 regulates the expression of genes associated with ovarian steroidogenesis. LRH-1 can be transported to transcriptionally inactive nuclear bodies after conjugation with small ubiquitin-related modifier (SUMO). In the present study, we investigated the effects of SUMO modification at five lysine residues of LRH-1 in rat granulosa cells. Lysine 289 could be conjugated with SUMO-1 in vitro, and the mutation K289R increased transcriptional activity of LRH-1, suggesting that SUMO conjugation is associated with transcription repression. Coexpression of SUMO-1 targets LRH-1 to the dot-like nuclear bodies, but the effect of lysine mutations on blocking subnuclear localization depended on the cell type. In COS-7 cells, mutation of either K173 or K289 prevented SUMO-1-mediated translocation of LRH-1 into nuclear bodies and also reduced the conjugation by SUMO-1, suggesting that K289 and K173 are two important sites involved in SUMO-1 modification. In granulosa cells, three or more altered lysine residues were required for nucleoplasm retention. This result suggests that multiple lysine residues are targets for SUMO conjugation in vivo and granulosa cells are more sensitive to SUMO-1-mediated LRH-1 localization to nuclear bodies. Nuclear body localization of LRH-1 was suppressed by forskolin and cholera toxin. Forskolin treatment obviously influences the expression of members involved in the SUMO pathway. The results obtained in the present study suggest that cAMP signaling could change the dynamic process of sumoylation and repress LRH-1 targeting to nuclear speckles in rat granulosa cells.

Regulation of cold signaling by sumoylation of ICE1

The small ubiquitin-related modifier (SUMO) E3 ligase SIZ1 is an ortholog of yeast and animal SIZ (SAP and Miz)/PIAS (protein inhibition of activated STAT) proteins, which function as transcriptional co-regulators either by facilitating SUMO conjugation to substrate proteins (sumoylation) or through other mechanisms that are sumoylation independent. SIZ/PIAS-type E3 ligases function in numerous eukaryotic biological processes, including regulation of organismal responses to environmental changes. This Addendum summarizes our recent paper in which it is established that the Arabidopsis E3 ligase SIZ1 mediates sumoylation of ICE1. SUMO conjugation to ICE facilitates ICE activity and stability that positively regulates CBF3/DREB1A-dependent cold signaling and freezing tolerance. Furthermore, sumoylated ICE1 represses MYB15, which is a negative regulator of CBF3/DREB1A and freezing tolerance.

SUMO: getting it on

Post-translational modification of cellular proteins by the SUMO (small ubiquitin-related modifier) is involved in numerous modes of regulation in widely different biological processes. In contrast with ubiquitination, SUMO conjugation is highly specific in terms of target lysine residues, but many aspects of substrate and lysine selection by the SUMO conjugating machinery are still poorly understood. SUMOylation events usually occur on the PsiKXE SUMO consensus motifs, which mediate binding to Ubc9 (ubiquitin-conjugating enzyme 9), the SUMO E2 conjugating enzyme. Although most, if not all, SUMO conjugations are catalysed by Ubc9, far from all PsiKXE tetrapeptides are modified, demonstrating a need for additional specificity determinants in SUMOylation. Recent results intimately link regulation of SUMOylation to other post-translational modifications, including phosphorylation and acetylation and reveal that certain lysine residues are marked for SUMOylation by negatively charged amino acid residues or phosphorylation events immediately downstream of the consensus site. In the present review, we explore the intriguing role of extended motifs in the regulation of SUMO conjugation.

Signalling pathways and the regulation of SUMO modification

The modification of proteins by SUMO (small ubiquitin-related modifier) conjugation is becoming increasingly recognized as an important regulatory event. Protein SUMOylation can control a whole range of activities, including subcellular localization, protein-protein interactions and enzymatic activity. However, the SUMOylation process can itself be controlled. In the present review, the mechanisms through which protein SUMOylation is regulated are discussed, with particular emphasis on the impact of signalling pathways. A major point of regulation of the SUMO pathway is through targeting the E3 ligases, and a number of different ways to achieve this have been identified. More generally, the MAPK (mitogen-activated protein kinase) pathways represent one way through which SUMOylation of specific proteins is controlled, by using molecular mechanisms that at least in part also function by modifying the activity of SUMO E3 ligases. Further intricacies in signalling pathway interactions are hinted at through the growing number of examples of cross-talk between different post-translational modifications and SUMO modification.

Regulation of the dual-function transcription factor Sp3 by SUMO

In eukaryotes, gene expression is controlled by a relatively small number of regulators. Post-translational modifications dramatically increase the functional possibilities of those regulators. Modification of many transcription factors and cofactors by SUMO (small ubiquitin-related modifier) correlates, in most cases, with inhibition of transcription. Recent studies suggest a model whereby SUMO conjugation to transcription factors promotes the recruitment of co-repressors through direct protein-protein interaction with the SUMO protein. HDACs (histone deacetylases) are important, but not exclusive, effectors of SUMO-mediated repression. Sp3 (specificity protein 3), a zinc-finger DNA-binding domain transcription factor, has the ability to both activate and repress transcription in a context-dependent manner. SUMOylation regulates the dual nature of Sp3 function. Current data suggest that Sp3 represses transcription in a SUMO-dependent manner but independent of HDACs. Recent studies to identify additional co-repressors associated with SUMO and further investigate regulated activity of Sp3 are providing a deeper understanding of SUMO-dependent mechanisms of transcriptional regulation.

Ubiquitin-dependent Proteolytic Control of SUMO Conjugates

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.

XSUMO‐1 is required for normal mesoderm induction and axis elongation during early Xenopus development

The small ubiquitin-related modifier (SUMO) is a member of the ubiquitin-like protein family, and SUMO conjugation (SUMOylation) resembles ubiquitination. Despite many SUMOylation target proteins being reported, the role of this system in vertebrate development remains unclear. We inhibited the function of Xenopus SUMO-1 (XSUMO-1) using a morpholino antisense oligo against XSUMO-1 (XSUMO-1-MO) to clarify the role of SUMOylation. XSUMO-1-MO inhibited normal axis formation in embryos and elongation of activin-treated animal caps. The expression of several mesoderm markers was reduced by XSUMO-1-MO. We measured activin-like activity by using a reporter construct containing a multimer of activin-responsive elements from the Goosecoid promoter, [DE(6x)Luc]. This assay showed that XSUMO-1-MO directly inhibited activin/nodal signaling. Furthermore, XSUMO-1-MO inhibited ectopic axis formation induced by XSmad2, and XSmad2/4 mRNA could not rescue the axis elongation defect induced by XSUMO-1-MO. These results suggested that XSUMO-1 is required for normal axis elongation, at least partly mediating activin/nodal signaling.