RING-type E3 Ubiquitin Ligase Research Articles

Anti-viral tetris: modulation of the innate anti-viral immune response by A20.

The A20 protein has emerged as an important negative regulator of Toll like receptor (TLR) and retinoic acid-inducible gene 1 (RIG-I)-mediated anti-viral signaling. A20 functions both as a RING-type E3 ubiquitin ligase and as a de-ubiquitinating enzyme. Nuclear factor kappa B (NF-kappaB) and interferon regulatory factor (IRF) pathways are targeted by A20 through mechanisms that appear to be both overlapping and distinct, resulting in the downregulation of interferon alpha/beta (IFNalpha/beta) production. This review specifically details the impact of A20 on the cytosolic RIG-I/MDA5 pathway, a process that is less understood than that of NF-kappaB but is essential for the regulation of the innate immune response to viral infection.

Natural variation in PTB1 regulates rice seed setting rate by controlling pollen tube growth

Grain number, panicle seed setting rate, panicle number and grain weight are the most important components of rice grain yield. To date, several genes related to grain weight, grain number and panicle number have been described in rice. However, no genes regulating the panicle seed setting rate have been functionally characterized. Here we show that the domestication-related POLLEN TUBE BLOCKED 1 (PTB1), a RING-type E3 ubiquitin ligase, positively regulates the rice panicle seed setting rate by promoting pollen tube growth. The natural variation in expression of PTB1 which is affected by the promoter haplotype and the environmental temperature, correlates with the rice panicle seed setting rate. Our results support the hypothesis that PTB1 is an important maternal sporophytic factor of pollen tube growth and a key modulator of the rice panicle seed setting rate. This finding has implications for the improvement of rice yield.

Arabidopsis ubiquitin ligase MIEL1 mediates degradation of the transcription factor MYB30 weakening plant defence

One of the most efficient plant resistance reactions to pathogen attack is the hypersensitive response, a form of programmed cell death at infection sites. The Arabidopsis transcription factor MYB30 is a positive regulator of hypersensitive cell death responses. Here we show that MIEL1 (MYB30-Interacting E3 Ligase1), an Arabidopsis RING-type E3 ubiquitin ligase that interacts with and ubiquitinates MYB30, leads to MYB30 proteasomal degradation and downregulation of its transcriptional activity. In non-infected plants, MIEL1 attenuates cell death and defence through degradation of MYB30. Following bacterial inoculation, repression of MIEL1 expression removes this negative regulation allowing sufficient MYB30 accumulation in the inoculated zone to trigger the hypersensitive response and restrict pathogen growth. Our work underlines the important role played by ubiquitination to control the hypersensitive response and highlights the sophisticated fine-tuning of plant responses to pathogen attack. Overall, this work emphasizes the importance of protein modification by ubiquitination during the regulation of transcriptional responses to stress in eukaryotic cells.

Aggregated Low-Density Lipoprotein Induces LRP1 Stabilization Through E3 Ubiquitin Ligase CHFR Downregulation in Human Vascular Smooth Muscle Cells

Low density lipoprotein retention and aggregation in the arterial intima are key processes in atherogenesis. Aggregated LDL (agLDL) is taken up through low-density lipoprotein receptor-related protein 1 (LRP1) by human vascular smooth muscle cells (VSMC). AgLDL increases LRP1 expression, at least in part, by downregulation of sterol regulatory element-binding proteins. It is unknown whether agLDL has some effect on the ubiquitin-proteasome system, and therefore on the LRP1 receptor turnover. The objective of this study was to analyze the effect of agLDL on the degradation of LRP1 by the ubiquitin-proteasome system in human VSMC. Human VSMC were isolated from the media of human coronary arteries. Ubiquitinylated LRP1 protein levels were significantly reduced in human VSMC exposed to agLDL (100 μg/mL) for 20 hours (agLDL: 3.70±0.44 a.u. versus control: 9.68±0.55 a.u). Studies performed with cycloheximide showed that agLDL prolongs the LRP1 protein half life. Pulse-chase analysis showed that LRP1 turnover rate is reduced in agLDL-exposed VSMC. Two-dimensional electrophoresis shows an alteration in the proteomic profile of a RING type E3 ubiquitin ligase, CHFR. Real-time PCR and Western blot analysis showed that agLDL (100 μg/mL) decreased the transcriptional and protein expression of CHFR. CHFR silencing increased VSMC, but not macrophage, LRP1 expression. However, CHFR silencing did not exert any effect on the classical low-density lipoprotein receptor protein levels. Furthermore, immunoprecipitation experiments demonstrated that the physical interaction between CHFR and LRP1 decreased in the presence of agLDL. Our results demonstrate that agLDL prolongs the half life of LRP1 by preventing the receptor ubiquitinylation, at least in part, through CHFR targeting. This mechanism seems to be specific for LRP1 and VSMC.

RING-finger type E3 ubiquitin ligase inhibitors as novel candidates for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) significantly affects quality of life. We recently cloned synoviolin, a RING-type E3 ubiquitin ligase implicated in the endoplasmic reticulum-associated degradation (ERAD) pathway. Synoviolin is highly expressed in rheumatoid synovial cells and may be involved in the pathogenesis of RA. Inhibition of synoviolin activity is a potentially useful therapeutic approach for the treatment of RA. We conducted a high-throughput screen of small molecules to find inhibitors of synoviolin autoubiquitination activity. We identified two classes of small molecules, named LS-101 and LS-102, which inhibited synoviolin activity. LS-102 selectively inhibited synoviolin enzymatic activity, while LS-101 inhibited a broad array of RING-type E3 ligases. Moreover, these inhibitors suppressed the proliferation of rheumatoid synovial cells, and significantly reduced the severity of disease in a mouse model of RA. Our results suggest that inhibition of synoviolin is a potentially useful approach in the treatment of RA.

Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase

Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by in vitro ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated in vitro, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed in vivo. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale.

Identification of a novel motif in MARCH1 that affects the conformation and the E3 ubiquitin ligase activity (65.10)

Abstract Some members of the MARCH family of RING-type E3 ubiquitin ligases target important players of immune responses. Of these, MARCH1 prevents the cell surface accumulation of MHC class II molecules and regulates antigen presentation in response to IL-10 in human primary monocytes (Thibodeau et. al., Eur JI 2008). Structure-function analysis of mouse MARCH1 showed the presence of two functional tyrosine-based endosomal sorting motifs in the C-terminal cytoplasmic part of the protein. We investigated the importance of the corresponding motifs in human MARCH1. Site-directed mutagenesis of these tyrosines did not reduce the activity of MARCH1 in transfected cells. However, a reporter molecule fused to the C-terminal cytoplasmic domain of MARCH1 was expressed less efficiently at the plasma membrane. A series of deletions were made in the C-terminal part of MARCH1 and a new functional motif was discovered. BRET experiments revealed that this motif is important for the spatial organisation of the two cytoplasmic domains of MARCH1. Interestingly, this motif is also present in others RING-type E3 ubiquitin ligases such as Parkin and its alteration is linked to the development of the disease in certain patients.

Regulation of glycogen phosphorylase by the malin‐laforin complex

Autosomal recessive mutations in genes encoding malin or laforin cause Lafora disease (LD), a progressive myoclonic epilepsy. Pathological aberrant glycogen accumulations in brain and various other organs are a hallmark of LD. Our group identified laforin as a novel glycogen phosphatase essential for maintaining glycogen in a soluble form. Malin is a RING‐type E3 ubiquitin ligase. Malin is known to ubiquitinate proteins involved in glycogen metabolism such as laforin, glycogen synthase, protein targeting to glycogen and glycogen debranching enzyme. However, except laforin, expression of these proteins is unchanged in malin knockout mice and thus malin's role in LD pathogenesis is still unclear. Glycogen phosphorylase and glycogen debranching enzyme are vital in catabolism of cellular glycogen. In this study, we aimed to discover substrate/s of malin and its function in LD pathogenesis. We employed immunoprecipitation, western analysis, immunocytochemistry and enzyme activity measurements. Our data uncovered glycogen phosphorylase as a novel substrate of malin. The expression of glycogen phosphorylase was decreased in liver and skeletal muscle tissues from malin knockout mice and glycogen phosphorylase expression was unchanged in tissues from laforin knockout mice. Similarly, overexpression of LD mutations of malin in HEK‐293 cells decreased glycogen phosphorylase expression. In addition, transient overexpression of malin in HEK‐ 293 cells increased the endogenous expression of glycogen phosphorylase. Taken together, our results have identified a novel substrate of malin. Further exploration into the mechanisms of this regulation will unravel insights in glycogen metabolism, physiological functions of malin‐laforin complex and will aid in betterment of LD pharmacotherapy.Supported by NIH grants R00NS061803, R01NS070899 and University of Kentucky College of Medicine startup funds.

The Ubiquitin E3 Ligase LOSS OF GDU2 Is Required for GLUTAMINE DUMPER1-Induced Amino Acid Secretion in Arabidopsis

Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.

Identification of a novel motif that affects the conformation and activity of the MARCH1 E3 ubiquitin ligase

MARCH1, a member of the membrane-associated RING-CH family of E3 ubiquitin ligases, regulates antigen presentation by downregulating the cell surface expression of Major Histocompatibility Complex class II and CD86 molecules. MARCH1 is a transmembrane protein that exposes both its N- and C-terminus to the cytoplasm. We have conducted a structure-function analysis of its two cytoplasmic tails to gain insights into the trafficking of MARCH1 in the endocytic pathway. Fusion of the N-terminal portion of MARCH1 to a type II transmembrane reporter molecule revealed that this cytoplasmic tail contains endosomal sorting motifs. The C-terminal domain also appears to contain intracellular sorting signals because it reduced surface expression of a type I transmembrane reporter molecule. Mutation of the two putative C-terminal tyrosine-based sorting signals did not affect the activity of human MARCH1; however, it did reduce its incorporation into exosomes. Moreover, site-directed mutagenesis pointed to a functional C-terminal 221VQNC224 sequence that affects the spatial organization of the two cytoplasmic regions. This motif is also found in other RING-type E3 ubiquitin ligases, such as parkin. Altogether, these findings highlight the complex regulation of MARCH1 trafficking in the endocytic pathway as well as the intricate interactions between its cytoplasmic tails.

Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

TRIM proteins and cancer

Emerging clinical evidence shows that the deregulation of ubiquitin-mediated degradation of oncogene products or tumour suppressors is likely to be involved in the aetiology of carcinomas and leukaemias. Recent studies have indicated that some members of the tripartite motif (TRIM) proteins (one of the subfamilies of the RING type E3 ubiquitin ligases) function as important regulators for carcinogenesis. This Review focuses on TRIM proteins that are involved in tumour development and progression.

RB1CC1 Protein Positively Regulates Transforming Growth Factor-β Signaling through the Modulation of Arkadia E3 Ubiquitin Ligase Activity

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia.

Interaction of an intracellular pentraxin with a BTB-Kelch protein is associated with ubiquitylation, aggregation and neuronal apoptosis

Neuronal pentraxin with chromo domain (NPCD) comprises a group of neuronally expressed pentraxins with both membrane and cytosolic isoforms; the functions of cytosolic NPCD isoforms are not clear. Here, we demonstrate that a cytosolic NPCD isoform selectively interacts with the BTB-Kelch protein Mayven/Kelch-like 2 (KLHL2), an actin-binding protein implicated in process outgrowth in oligodendrocytes. The KLHL2–NPCD interaction was identified by a yeast two-hybrid screen and confirmed through colocalization and co-immunoprecipitation studies. Truncation analysis indicates that the Kelch domains of KLHL2 interact with the pentraxin domain of NPCD. NPCD forms protein inclusion bodies (aggresomes) when overexpressed in tissue culture cells, KLHL2 localizes to these aggresomes, and overexpression of KLHL2 increases NPCD aggresome formation. Since other members of the BTB-Kelch family can act as Cullin–RING type E3 ubiquitin ligases, we tested the potential role of KLHL2 as a ubiquitin ligase for NPCD. We found that KLHL2 interacts selectively with Cullin 3, a key component of BTB-Kelch ubiquitin ligase complexes. Further, overexpression of KLHL2 promotes NPCD ubiquitylation. Together, these results suggest a novel E3 ubiquitin ligase function of KLHL2, with NPCD as a substrate. As the formation of aggresomes is often associated with protein aggregation in neurodegenerative diseases, we tested the effects of NPCD overexpression and KLHL2 coexpression on neuronal viability. Overexpression of NPCD in hippocampal neurons led to cell death and apoptosis; this effect was exacerbated by KLHL2 co-expression. Our findings implicate KLHL2 in ubiquitin ligase activity, and suggest potential roles of NPCD and KLHL2 in neurodegeneration.

Identification of a novel motif important for the activity of ring-type E3 ligases using MARCH1 as a model (100.52)

Abstract The MARCH family of proteins are RING-CH type E3 ubiquitin ligases responsible for the ubiquitination and endocytosis of cell surface proteins related to immune response. We are particularty interested in MARCH1 and 8, two members of the family that are known to downregulate the cell surface expression of MHC II, CD86 , TfR and CD95. Canonical sorting motifs in the cytoplasmic portions of murine MARCH1 and human MARCH8 were identified and reported by others to be necessary for the function. We hypothesized that such motifs in human MARCH1 are important for it’s function. Methods: We used co-immunoprecipitations and FRET to monitor interaction of MARCH1 with various proteins. Protein expression, function and localization were determined by flow cytometry, confocal microscopy and western blotting. Results: Our results showed the presence of functional sorting motifs in both N- and C-terminal cytoplasmic portions of human MARCH1. However, these signals are not essential for the activity of MARCH1. We used a series of mutants to identify functional regions on MARCH1. Our results point to a novel motif. Interestingly, that motif is found in many other proteins and appears to be important for the function of other RING-type E3 ligases. Conclusions: Our results confirmed the presence of sorting motifs in MARCH1 and highlight important species-specific functional domains. The newly identified domain of MARCH1 will shed light on the structure of RING-type E3 ubiquitin ligases.

The TRAF-Interacting Protein (TRIP) Is a Regulator of Keratinocyte Proliferation

The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis factor-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in keratinocytes. TRIP mRNA expression was strongly downregulated in primary human keratinocytes undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-12-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative keratinocytes suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. Keratinocytes undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused growth arrest in the G1/S phase. Keratinocytes with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD keratinocytes, indicating that NF-κB activity in keratinocytes is not regulated by TRIP. TRIP expression was increased by ∼2-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to keratinocyte proliferation.

The myostatin‐induced E3 ubiquitin ligase RNF13 negatively regulates the proliferation of chicken myoblasts

The ubiquitin ligase RING finger protein 13 gene (RNF13) was first identified in a screen for genes whose expression is regulated by myostatin in chicken fetal myoblasts. In this study, we demonstrate that the RNF13 gene is broadly expressed in many chicken tissues. The expression of RNF13 gradually decreases during skeletal myogenesis, and myostatin up-regulates RNF13 expression at both the transcriptional and translational levels. Interestingly, ectopic expression of RNF13 inhibits cell proliferation and suppresses the expression of the myogenic genes MyoD and Caveolin-3 in muscle cells. Moreover, recently, we have reported that RNF13 is a RING-type E3 ubiquitin ligase. In this report, we provide experimental evidence to show that mutations disrupting the RING finger abolish the growth-suppressive activity of RNF13, indicating that its E3 ligase activity is required for the inhibition of cell proliferation. Taken together, our findings show that RNF13 functions as an E3 ubiquitin ligase to negatively regulate cell proliferation.

BCA2/Rabring7 Promotes Tetherin-Dependent HIV-1 Restriction

Host cell factors can either positively or negatively regulate the assembly and egress of HIV-1 particles from infected cells. Recent reports have identified a previously uncharacterized transmembrane protein, tetherin/CD317/BST-2, as a crucial host restriction factor that acts during a late budding step in HIV-1 replication by inhibiting viral particle release. Although tetherin has been shown to promote the retention of nascent viral particles on the host cell surface, the precise molecular mechanisms that occur during and after these tethering events remain largely unknown. We here report that a RING-type E3 ubiquitin ligase, BCA2 (Breast cancer-associated gene 2; also called Rabring7, ZNF364 or RNF115), is a novel tetherin-interacting host protein that facilitates the restriction of HIV-1 particle production in tetherin-positive cells. The expression of human BCA2 in “tetherin-positive” HeLa, but not in “tetherin-negative” HOS cells, resulted in a strong restriction of HIV-1 particle production. Upon the expression of tetherin in HOS cells, BCA2 was capable of inhibiting viral particle production as in HeLa cells. The targeted depletion of endogenous BCA2 by RNA interference (RNAi) in HeLa cells reduced the intracellular accumulation of viral particles, which were nevertheless retained on the plasma membrane. BCA2 was also found to facilitate the internalization of HIV-1 virions into CD63+ intracellular vesicles leading to their lysosomal degradation. These results indicate that BCA2 accelerates the internalization and degradation of viral particles following their tethering to the cell surface and is a co-factor or enhancer for the tetherin-dependent restriction of HIV-1 release from infected cells.

SH3 Domains from a Subset of BAR Proteins Define a Ubl-Binding Domain and Implicate Parkin in Synaptic Ubiquitination

Mutations in the parkin gene are responsible for a common inherited form of Parkinson's disease (PD). Parkin is a RING-type E3 ubiquitin ligase with an N-terminal ubiquitin-like domain (Ubl). We report here that the parkin Ubl binds SH3 domains from endocytic BAR proteins such as endophilin-A with an affinity comparable to proline-rich domains (PRDs) from well-established SH3 partners. The NMR structure of the Ubl-SH3 complex identifies the PaRK extension, a unique C-terminal motif in the parkin Ubl required for SH3 binding and for parkin-mediated ubiquitination of endophilin-A in vitro. In nerve terminals, conditions that promote phosphorylation enhance the interaction between parkin and endophilin-A and increase the levels of ubiquitinated proteins within PRD-associated synaptic protein complexes in wild-type but not parkin knockout brain. The findings identify a pathway for the recruitment of synaptic substrates to parkin with the potential to explain the defects in synaptic transmission observed in recessive forms of PD.

GmRFP1 encodes a previously unknown RING-type E3 ubiquitin ligase in Soybean (Glycine max)

RING-finger proteins with E3 ubiquitin ligase activity play important roles in the regulation of plant growth and development. In this study, a cDNA clone encoding a novel RING-finger protein, designated as GmRFP1, was isolated and characterized from soybean. GmRFP1 was an intronless gene encoding a predicted protein product of 392 amino acid residues with a molecular mass of ~43 kDa. The protein contained a RING-H2 motif and an N-terminal transmembrane domain. The transcript was observed in all detected organs and was up-regulated by abscisic acid (ABA) and salt stress, but down-regulated by cold and drought treatments. We further expressed and purified both wild type and mutant version of GmRFP1 in E. coli. In vitro assays showed that the purified GmRFP1 induced the formation of polyubiquitin chains while mutation within the RING-finger region abolished the ubiquitination activity. These findings suggest that GmRFP1 is a previously unknown E3 ubiquitin ligase in soybean and that the RING domain is required for its activity. It may play unappreciated roles in ABA signaling and stress responses via mediating the ubiquitination and degradation of target proteins through the ubiquitin-proteasome pathway.