Regulation of DNA topoisomerase IIα stability by the ECV ubiquitin ligase complex

In this study, we attempted to elucidate the E3 ubiquitin ligase for topo IIα. When cullins and VHL were ectopically expressed in HT1080 and HEK293T cells, topo IIα was degraded most prominently in cullin 2- and VHL-expressing cells. Cullin 2 and the β domain (aa 114-123) of VHL, a subunit of the ECV (Elongin B/C-cullin 2-VHL protein) complex, specifically interact with the ATPase domain of topo IIα. We identified that topo IIα associated with endogenous Elongin C. In HT1080 cells co-transfected with deletion mutants of topo IIα GRDD (glucose-regulated destruction domain) and VHL, topo IIα was degraded by VHL expression. These results demonstrate that ECV acts as E3 ubiquitin ligase targeting GRDD-independent topo IIα to the ubiquitin-proteasome pathway.     

Regulation of PTEN degradation and NEDD4–1 E3 ligase activity by Numb

The critical tumor suppressor PTEN is regulated by numerous post-translational modifications including phosphorylation, acetylation and ubiquitination. Ubiquitination of PTEN was reported to control both PTEN stability and nuclear localization. Notably, the HECT E3-ligase NEDD4–1 was identified as the ubiquitin ligase for PTEN, mediating its degradation and down-stream events. However, the mechanisms how NEDD4–1 is regulated by up-stream signaling pathways or interaction with other proteins in promoting PTEN degradation remain largely unclear. In the present study, we identified that the adaptor protein Numb, which is demonstrated to be a novel binding partner of NEDD4–1, plays important roles in controlling PTEN ubiquitination through regulating NEDD4–1 activity and the association between PTEN and NEDD4–1. Furthermore, we provided data to show that Numb regulates cell proliferation and glucose metabolism in a PTEN-dependent manner. Overall, our study revealed a novel regulation of the well-documented NEDD4–1/PTEN pathway and its oncogenic behavior.     

Is the Rsp5 ubiquitin ligase involved in the regulation of ribophagy?

Under nutrient limiting conditions, cytoplasmic components are randomly sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for degradation and recycling. In the last few years, however, it has been observed that several cytoplasmic components such as organelles, pathogens or specific protein complexes can also be selectively targeted for degradation by autophagy-related pathways (reviewed in ref. 1). We have recently shown that in S. cerevisiae, mature ribosomes are subject to such selective degradation by autophagy under starvation conditions, in a process that we termed 'ribophagy.'(2) By genetic screening, we found that selective degradation of 60S large ribosomal subunits depends on the ubiquitin protease Ubp3 and its cofactor Bre5, implying that ribophagy is regulated by ubiquitin-dependent steps. Interestingly, several ubiquitinated proteins accumulate in ribosome fractions isolated from ubp3Delta cells, suggesting that the regulation of ribophagy by ubiquitin may be direct. Here we present data on a potential role of the ubiquitin ligase Rsp5 as a positive regulator of ribophagy, and discuss the possible involvement of ubiquitin as a signaling molecule in this process.     

Is the Rsp5 ubiquitin ligase involved in the regulation of ribophagy?

Under nutrient limiting conditions, cytoplasmic components are randomly sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for degradation and recycling. In the last few years, however, it has been observed that several cytoplasmic components such as organelles, pathogens or specific protein complexes can also be selectively targeted for degradation by autophagy-related pathways (reviewed in reference 1). We have recently shown that in S. cerevisiae, mature ribosomes are subject to such selective degradation by autophagy under starvation conditions, in a process that we termed ‘ribophagy’.² By genetic screening, we found that selective degradation of 60S large ribosomal subunits depends on the ubiquitin protease Ubp3 and its cofactor Bre5, implying that ribophagy is regulated by ubiquitin-dependent steps. Interestingly, several ubiquitinated proteins accumulate in ribosome fractions isolated from ubp3? cells, suggesting that the regulation of ribophagy by ubiquitin may be direct. Here we present data on a potential role of the ubiquitin ligase Rsp5 as a positive regulator of ribophagy, and discuss the possible involvement of ubiquitin as a signaling molecule in this process.

Addendum to: Kraft C, Deplazes A, Sohrmann M, Peter M. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat Cell Biol 2008; 10:602-10.     

Identification and characterization of the epithelial polarity receptor ”Frizzled” in Hydra vulgaris

The Wnt signaling pathway plays an important role in the specification of cell patterning during development in many species. Here we report the isolation and characterization of a putative Wnt receptor, Frizzled, in Hydra vulgaris. Analysis of the amino acid sequence of Frizzled in hydra reveals that this receptor contains many strong sequence similarities to other known Frizzled receptors. Hydra divergence is estimated to have occurred about one billion years ago; thus comparison of the Frizzled sequence of hydra with that of other species is likely to provide important information on the structure and function of those highly conserved regions. Northern and Southern blotting reveal that the Frizzled receptor in hydra has a 2.34-kb message size, and that it is encoded by a single gene. In situ hybridization using hydra frizzled as a probe reveals that the receptor message is restricted to the endoderm in adult hydra. This distribution supports the hypothesis that the Frizzled receptor is functioning in a pathway that controls cell differentiation in hydra. Received: 24 September 1999 / Accepted: 7 December 1999     

Stimulation of duodenal epithelial HCO3− transport in the guinea pig and cat by luminal prostaglandin E2

Segments of guinea pig or cat duodenum distal to the Brunner gland containing area and devoid of bile or pancreatic secretions were cannulated in situ. The unbuffered luminal solution was gassed with 100% O₂ or N₂ and HCO₃⁻ transport titrated at pH 7.40 or 8.00 with solutions containing HCl. Cat duodenum transported HCO₃⁻ at a greater rate (∼17μeq, cm⁻¹, h⁻¹) than did jejunum in the same animals (∼5μeq, cm⁻¹, h⁻¹) and also developed a greater transmucosal electrical potential difference. Luminal application of PGE₂ (1 – 12 μM) in cat duodenum increased HCO₃⁻ transport and the potential difference. HCO₃⁻ transport by guinea pig duodenum (∼27 μeq, cm⁻¹, h⁻¹) was increased by luminal PGE₂ only in animals where transport had been inhibited by pretreatment with aspirin (30 mg/kg intravenously). Exposure of the cat duodenal lumen to HCl (1 – 25 mM, 5 min) stimulated HCO₃⁻ transport and continuous exposure of duodenum in the guinea pig to acid discharged from the stomach may increase endogenous prostaglandin concentrations, resulting in an apparent lack of effect of exogenous prostaglandins. The present results and previous similar findings in amphibians in vitro suggest that surface epithelial transport of HCO₃⁻ protects duodenal mucosa against acid.     

SCFFBXL¹⁵ regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1

Smad ubiquitination regulatory factor 1 (Smurf1), an homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligase, performs a crucial role in the regulation of the bone morphogenetic protein (BMP) signalling pathway in both embryonic development and bone remodelling. How the stability and activity of Smurf1 are negatively regulated remains largely unclear. Here, we report that F-box and LRR domain-containing protein 15 (FBXL15), an F-box protein of the FBXL family, forms an Skp1-Cullin1-F-box protein-Roc1 (SCF)(FBXL15) ubiquitin ligase complex and targets Smurf1 for ubiquitination and proteasomal degradation. FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of the Smurf1 HECT domain and promotes the ubiquitination of Smurf1 on K355 and K357 within the WW-HECT linker region. In this way, FBXL15 positively regulates BMP signalling in mammalian cells. Knockdown of fbxl15 expression in zebrafish embryos by specific antisense morpholinos causes embryonic dorsalization phenocoping BMP-deficient mutants. Injection of FBXL15 siRNAs into rat bone tissues leads to a significant loss of bone mass and decrease in bone mineral density. Collectively, our results demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signalling during embryonic development and adult bone formation.     

REGγ proteasome mediates degradation of the ubiquitin ligase Smurf1

The ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) targets many proteins including Smad1/5 for ubiquitin-dependent proteasomal degradation. However, how Smurf1 is degraded remains unclear. Here we show that REGγ, an activator for the 20S proteasome-mediated protein degradation, interacts with Smurf1 and mediates its degradation. We provide evidence that depletion of REGγ stabilizes Smurf1 whereas overexpression of REGγ promotes the degradation of Smurf1. Interestingly both Smurf2 and Smurf1 are destabilized by the REGγ proteasome while the other members of Neural precursor cell-expressed developmentally downregulated gene 4 family were not affected. More importantly, we found that the REGγ proteasome-mediated degradation of Smurf1 results in degradation of Smad5. These findings reveal that the REGγ-proteasome targets a ubiquitin ligase for protein degradation.

Structured summary

MINT-7894509: CKIP (uniprotkb:Q53GL0) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull down (MI:0096)MINT-7894494: REG gamma (uniprotkb:P61289) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull down (MI:0096)MINT-7894523, MINT-7894543, MINT-7894481: REG gamma (uniprotkb:P61289) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag coimmunoprecipitation (MI:0007)MINT-7894558: Smurf1 (uniprotkb:Q9HCE7) physically interacts (MI:0915) with REG gamma (uniprotkb:P61289) by two hybrid (MI:0018)     

Characterization of the interaction of full-length HIV-1 Vif protein with its key regulator CBFβ and CRL5 E3 ubiquitin ligase components

Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.     

p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility

Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8–amino acid motif (533–541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41–amino acid region containing 14-3-3– and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER–mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER–mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER–mitochondria association and mitochondria motility.     

Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3β Homologue,Shaggy

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals.     

The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation

Background

The nuclear factor κB (NF-κB) family members regulate several biological processes as cell proliferation and differentiation, inflammation, immunity and tumor progression. Ubiquitination plays a key role in NF-κB activation and the ubiquitylated transmitters of the NF-κB signaling cascade accumulate in close proximity to endomembranes.

Findings

We performed an unbiased siRNA library screen targeting the 46 E3 ubiquitin ligases bearing transmembrane domains to uncover new modulators of NF-κB activation, using tumor necrosis factor–α (TNF-α) receptor (TNFR) stimulation as a model. We report here the identification of a new Golgi Apparatus-resident protein, RNF121, as an enhancer of NF-κB promoter activity through the catalytic function of its RING domain. From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process. However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex. Finally, we discovered that RNF121 acts as a broad regulator of NF-κB signaling since its silencing also dampens NF-κB activation following stimulation of Toll-Like Receptors (TLRs), Nod-Like Receptors (NLRs), RIG-I-Like Receptors (RLRs) or after DNA damages.

Conclusions

These results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

     

Role of β-TrCP ubiquitin ligase receptor in UVB mediated responses in skin

Skin cancers are the most common cancers in the United States. Exposure to UVB radiation is a major risk factor for skin cancer induction. SCF^β-TrCP E3 ubiquitin ligase has been found to be involved in cell cycle, cell proliferation and transformation. Aberrant up-regulation of beta-transducin repeats-containing proteins (β-TrCP) is often found in cancer cell lines and primary tumors. We have previously demonstrated that β-TrCP2 is over-expressed in chemically induced mouse skin tumors [1]. Various cellular stress stimuli, including UVB, induce an increase in β-TrCP1 mRNA and protein levels in human cells [2]. We have previously shown that inhibition of β-TrCP function, by induction of dominant negative β-TrCP2 (β-TrCP2^ΔF), in vitro in hTERT immortalized normal keratinocytes, results in increase in UVB induced apoptosis [3]. We have generated transgenic mice with inducible, selective expression of dominant negative β-TrCP2 in epidermis with the Keratin 5 promoter (K5-rTA × TRE-HA-β-TrCP^ΔF). Here we report that inhibition of β-TrCP function in mouse epidermis results in decrease in UVB-induced edema, hyperplasia, and inflammatory response and increment in UVB-induced apoptosis in skin. Our results suggest that β-TrCP may be an essential player in UVB induced responses in skin and can be a potential therapeutic target for skin cancer.