RING-box Protein Research Articles

Endoplasmic Reticulum Stress Induces a Caspase-dependent N-terminal Cleavage of RBX1 Protein in B Cells

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.

Induction of autophagy and senescence by knockdown of ROC1 E3 ubiquitin ligase to suppress the growth of liver cancer cells

Regulator of Cullins-1 (ROC1) or RING box protein-1 (RBX1) is an essential RING component of Cullin-RING ligase (CRL). Our previous studies showed that ROC1 is required for the growth of several cancer cell lines while ROC1 siRNA silencing inactivates CRL, leading to cell cycle arrest, cell senescence and/or apoptosis. However, it is completely unknown whether ROC1 knockdown triggers autophagic response by inactivating CRL. Moreover, the role of ROC1 in liver cancer remains elusive. In this study, we reported that ROC1 knockdown significantly inhibited the growth of liver cancer cells by sequentially and independently inducing autophagy and p21-dependent cell senescence. Mechanism analysis revealed that ROC1 silencing triggered autophagy by inhibition of mammalian target of rapamycin (mTOR) activity due to accumulation of mTOR-inhibitory protein Deptor, a substrate of CRL. Consistently, Deptor knockdown significantly blocked autophagy response upon ROC1 silencing. Biologically, autophagy response upon ROC1 silencing was a survival signal, and blockage of autophagy pathway sensitized cancer cells to apoptosis. Finally, we demonstrated that ROC1 was overexpressed in hepatocellular carcinomas, which is associated with poor prognosis of liver cancer patients. These findings suggest that ROC1 is an appealing drug target for liver cancer and provide a proof-of-concept evidence for a novel drug combination of ROC1 inhibitor and an autophagy inhibitor for effective treatment of liver cancer by enhancing apoptosis.

The Predicted Molecular Weight of Nrf2: It Is What It Is Not

The Predicted Molecular Weight of Nrf2: It Is What It Is Not

Selective Recruitment of an E2∼Ubiquitin Complex by an E3 Ubiquitin Ligase

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40-108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12-39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.

Vif hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host's antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β-Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.

RBX1 (RING Box Protein 1) E3 Ubiquitin Ligase Is Required for Genomic Integrity by Modulating DNA Replication Licensing Proteins

RBX1 (RING box protein 1), also known as ROC1 (Regulator of Cullin 1), is an essential component of SCF (Skp1/Cullins/F-box) E3 ubiquitin ligases, which target diverse proteins for proteasome-mediated degradation. Our recent study showed that RBX1 silencing triggered a DNA damage response (DDR) leading to G(2)-M arrest, senescence, and apoptosis, with the mechanism remaining elusive. Here, we show that, in human cancer cells, RBX1 silencing causes the accumulation of DNA replication licensing proteins CDT1 and ORC1, leading to DNA double-strand breaks, DDR, G(2) arrest, and, eventually, aneuploidy. Whereas CHK1 activation by RBX1 silencing is responsible for the G(2) arrest, enhanced DNA damage renders cancer cells more sensitive to radiation. In Caenorhabditis elegans, RBX-1 silencing causes CDT-1 accumulation, triggering DDR in intestinal cells, which is largely abrogated by simultaneous CDT-1 silencing. RBX-1 silencing also induces lethality during development of embryos and in adulthood. Thus, RBX1 E3 ligase is essential for the maintenance of mammalian genome integrity and the proper development and viability in C. elegans.

Abstract A14: DNA alterations to the Cullin-3/Ring box protein-1 E3 ubiquitin ligase complex represent a novel mechanism of NF-κB activation in lung cancer

Abstract Background: Nuclear factor kappa B (NF-κB) signaling is essential for lung cancer development, and therefore, may serve as a target for intervention. However, the genetic mechanisms responsible for its activation are not fully understood. Kelch-like ECH-associated protein 1 (KEAP1) binds protein substrates to the Cullin-3 (CUL3)/Ring box protein-1 (RBX1) E3 ubiquitin ligase complex where ubiquitination signals substrates for proteosomal degradation. Recently, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), an activator of NF-κB, was shown to be a substrate of KEAP1, implicating KEAP1's involvement in regulating NF-κB signaling. Loss of function of KEAP1 leads to IKKβ accumulation and NF-κB activation. We hypothesized that DNA deletions of the other E3 complex components (CUL3 and RBX1) are frequent alterations that disrupt complex function and contribute to NF-κB activation in lung cancer. Methods: We screened DNA copy number profiles of 261 non-small cell lung cancer (NSCLC) tumors for DNA alterations at the KEAP1, CUL3, RBX1, and IKK loci. Profiles were generated by array comparative genomic hybridization on the SMRT array (sub-megabase resolution tiling) platform. We also analyzed mRNA expression of these genes and 9 well annotated NF-κB target genes, using gene expression profiles generated with Agilent gene expression microarrays for a subset (n=48) of the tumors. Results: Our investigation of genetic disruption to the E3 ubiquitin ligase complex components revealed 54% of tumors harbored DNA copy number loss of at least one complex component (KEAP1, CUL3, or RBX1) or gain of IKKβ. Moreover, at the expression level, 81% of tumors analyzed had aberrant expression of one of these genes (underexpression of complex components or overexpression of IKKβ). Interestingly, the copy number alterations identified appeared to segregate with adenocarcinoma (AC) or squamous cell carcinoma (SCC) histology; KEAP1 loss was more prevalent in AC while CUL3 loss and IKKβ gain were more frequent in SCC. When NF-κB target gene expression was analyzed, we observed higher expression of 5/9 genes in tumors with underexpression of an E3 ubiquitin ligase complex component relative to matched non-malignant tissue from the same individual. Conclusions: The presence and strikingly high frequency of genetic disruption and aberrant expression of the E3 ubiquitin ligase complex components (KEAP1, CUL3, and RBX1) revealed in this study provides evidence of its importance in lung cancer. These data suggest that DNA level alterations to this complex may represent a novel mechanism of NF-κB activation in lung cancer. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A14.

Small RING Finger Proteins RBX1 and RBX2 of SCF E3 Ubiquitin Ligases: The Role in Cancer and as Cancer Targets

The SCF (Skp1-cullin-F-box proteins), also known as CRL (cullin-based RING ligase), is the largest family of E3 ubiquitin ligases that mediate approximately 20% ubiquitinated protein substrates for 26S proteasome degradation. Through promoting timely degradation of many key regulatory proteins, SCF E3 ligase controls numerous cellular processes; its dysfunction contributes to a number of human diseases, including cancer. The RING component of SCF complex consists of 2 family members, RBX1 (RING box protein 1), also known as ROC1 (regulator of cullins), and RBX2/ROC2 (also known as SAG [sensitive to apoptosis gene]), both of which are essential for the catalytic activity of SCF. RBX1 and RBX2 are evolutionarily conserved from yeast to humans and play an essential role during mouse embryonic development. Moreover, RBX1 and RBX2 are both overexpressed in multiple human cancer tissues and required for the growth and survival of cancer cells. In this review, we will discuss the similarities and differences between 2 RING family members, their regulation of SCF E3 ligase activity, and their role in development, cancer cell survival, and skin carcinogenesis, along with a brief discussion of RBX-SCF E3 ligases as the cancer targets and a recently discovered small molecule inhibitor of SCF E3 ligases as a novel class of anticancer drugs.

ROC1/RBX1 E3 Ubiquitin Ligase Silencing Suppresses Tumor Cell Growth via Sequential Induction of G2-M Arrest, Apoptosis, and Senescence

Regulator of Cullins-1 (ROC1) or Ring Box Protein-1 (RBX1) is a RING component of SCF (Skp-1, cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting a variety of substrates for degradation. However, little is known about the role of ROC1 in human cancer. Here, we report that ROC1 is ubiquitously overexpressed in primary human tumor tissues and human cancer cell lines. ROC1 silencing by siRNA significantly inhibited the growth of multiple human cancer cell lines via induction of senescence and apoptosis as well as G(2)-M arrest. Senescence induction is coupled with DNA damage in p53/p21- and p16/pRB-independent manners. Apoptosis is associated with accumulation of Puma and reduction of Bcl-2, Mcl-1, and survivin; and G(2)-M arrest is associated with accumulation of 14-3-3sigma and elimination of cyclin B1 and Cdc2. In U87 glioblastoma cells, these phenotypic changes occur sequentially upon ROC1 silencing, starting with G(2)-M arrest, followed by apoptosis and senescence. Thus, ROC1 silencing triggers multiple death and growth arrest pathways to effectively suppress tumor cell growth, suggesting that ROC1 may serve as a potential anticancer target.

Cullin3-Based Polyubiquitination and p62-Dependent Aggregation of Caspase-8 Mediate Extrinsic Apoptosis Signaling

Cell-surface death receptors such as DR4 and DR5 trigger apoptosis through a death-inducing signaling complex (DISC) that recruits the apical protease caspase-8. Apoptosis commitment requires efficient activation and autocatalytic release of caspase-8 into the cytoplasm to engage executioner caspases. While DISC recruitment initiates caspase-8 stimulation, full activation of the protease depends on further molecular aggregation events that are not fully understood. Here, we show that death receptor ligation induces polyubiquitination of caspase-8, through a previously unknown interaction of the DISC with a cullin3 (CUL3)-based E3 ligase. CUL3-mediated caspase-8 polyubiquitination required the RING box protein RBX1, whereas the deubiquitinase A20 reversed this modification. The ubiquitin-binding protein p62/sequestosome-1 promoted aggregation of CUL3-modified caspase-8 within p62-dependent foci, leading to full activation and processing of the enzyme and driving commitment to cell death. These results identify a mechanism that positively controls apoptosis signaling by polyubiquitination and aggregation of a key initiator caspase.

RBX1/ROC1 disruption results in early embryonic lethality due to proliferation failure, partially rescued by simultaneous loss of p27

RBX1 (RING box protein-1) or ROC1 (regulator of cullins-1) is the RING component of SCF (Skp1, Cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting various substrates for degradation. However, the in vivo physiological function of RBX1 remains uncharacterized. Here, we show that a gene trap disruption of mouse Rbx1 causes embryonic lethality at embryonic day (E)7.5, mainly due to a failure in proliferation; p27, a cyclin dependent kinase inhibitor, normally undetectable in the early embryos, accumulates at high levels in the absence of Rbx1. Although mice heterozygous for the Rbx1 gene trap appear viable and fertile without obvious abnormalities, the Rbx1(+/Gt) MEFs do show retarded growth with G1 arrest and p27 accumulation. Simultaneous loss of p27 extended the life span of Rbx1(Gt/Gt) embryos from E6.5 to E9.5, indicating that p27-mediated cell cycle inhibition contributes to the early embryonic lethality in the Rbx1-deficient embryos. Our study demonstrates that the in vivo physiological function of RBX1 is to ensure cell proliferation by preventing p27 accumulation during the early stage of embryonic development.

Lessons from Fungal F-Box Proteins

Lessons from Fungal F-Box Proteins

SAG/ROC2/RBX2 E3 ligase promotes UVB-induced skin hyperplasia, but not skin tumors, by simultaneously targeting c-Jun/AP-1 and p27

Sensitive to apoptosis gene (SAG)/regulator of cullins-2/RING box protein 2 is a stress-responsive RING component of Skp-1/Cullins/F-box protein E3 ubiquitin ligase. When overexpressed, SAG inhibits apoptosis induced by reactive oxygen species or hypoxia. Here, we report that SAG overexpression inhibits ultraviolet (UV) B-induced apoptosis in mouse JB6 epidermal cells. Using a transgenic mouse model, in which SAG expression was targeted primarily to epidermis by a K14 promoter, we showed that, at the early stage of UVB skin carcinogenesis (10 weeks post-UVB exposure), c-Jun, p27, p53, c-Fos and cyclin D1 were strongly induced. While having no effect on UVB-induced p53, c-Fos and cyclin D1, SAG-transgenic expression reduced the levels of c-Jun and p27 and inhibited AP-1 activity. The net outcome of SAG-mediated inhibition of c-Jun/AP-1 (pro-tumor promotion) and of p27 (antiproliferation) increased skin hyperplasia, with no apparent effect on apoptosis, as evidenced by increased skin thickness, and increased rate of DNA synthesis, but hardly any apoptosis. Although skin hyperplasia was promoted, SAG-transgenic expression had no significant effect on tumor formation in the later stage of UVB carcinogenesis. Thus, by simultaneously targeting c-Jun and p27, SAG accelerates UVB-induced skin hyperplasia, but not carcinogenesis.

RING-box protein 2

RING-box protein 2

Schistosoma mansoni: Heterologous complementation of a yeast null mutant by SmRbx, a protein similar to a RING box protein involved in ubiquitination

The SCF (Skp1–Cul1–F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1− yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxΔ24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.

Promotion of S-phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: association with inhibition of p27 accumulation.

The sensitive-to-apoptosis gene (SAG) was initially identified as a redox-inducible, apoptosis-protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S-phase entry as determined by [(3)H]-thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek-1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin-dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG-induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG-overexpressing cells, serum starvation induced 10- to 18-fold accumulation of p27 in control Rhek-1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5-fold) in SAG-infected cells. Inhibition of p27 accumulation was also observed in stably SAG-overexpressing SY5Y cells. Significantly, SAG-associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F-box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG-overexpressing cells grown under serum starvation. Thus, SAG-induced growth with serum withdrawal appears to be associated with SAG-mediated p27 degradation. Mol. Carcinog. 30:37-46, 2001.

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation.

In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox agents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability. Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants. Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA content. Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase. Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal. Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins. Oncogene (2000) 19, 2855 - 2866