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At least 667 records · Page 37Linked to original sources
PubMed2006

Phosphorylation-dependent control of Pc2 SUMO E3 ligase activity by its substrate protein HIPK2.

Sumoylation serves to control key cellular functions, but the regulation of SUMO E3 ligase activity is largely unknown. Here we show that the polycomb group protein Pc2 binds to and colocalizes with homeodomain interacting protein kinase 2 (HIPK2) and serves as a SUMO E3 ligase for this kinase. DNA damage-induced HIPK2 directly phosphorylates Pc2 at multiple sites, which in turn controls Pc2 sumoylation and intranuclear localization. Inducible phosphorylation of Pc2 at threonine 495 is required for its ability to increase HIPK2 sumoylation in response to DNA damage, thereby establishing an autoregulatory feedback loop between a SUMO substrate and its cognate E3 ligase. Sumoylation enhances the ability of HIPK2 to mediate transcriptional repression, thus providing a mechanistic link for DNA damage-induced transcriptional silencing.

Carrier Proteins↗
PubMed2006

Identification of N-glycan-binding proteins for E3 ubiquitin ligases.

N-glycans serve as a degradation signal by the SCF(Fbx2) ubiquitin ligase complex in the cytosol. Fbx2, an F-box protein, binds specifically to proteins attached with N-linked high-mannose type oligosaccharides, and subsequently contributes to ubiquitination of glycoproteins. Pre-integrin beta1 is identified as one of the Fbx2 targets. These two proteins bind in the cytosol after inhibition of the proteasome. These results indicate that SCF(Fbx2) ubiquitinates N-linked glycoproteins, which are translocated from the endoplasmic reticulum to the cytosol by the quality control mechanism. This chapter describes methods, including a binding protein assay for N-glycans, a ubiquitination assay for N-linked glycoproteins with SCF(Fbx2) ubiquitin ligase complex, an overlay assay for the detection of Fbx2 binding proteins, and a pull-down assay for the interaction between Fbx2 and N-linked glycoproteins, used to identify N-glycan-binding proteins for E3 ubiquitin ligases.

Animals↗
PubMed2003

Kinetoplastid RNA editing ligases: complex association, characterization, and substrate requirements.

RNA editing processes kinetoplastid mitochondrial transcripts post-transcriptionally by inserting and deleting uridylates (Us) to produce functional mRNAs. The activities of the RNA ligases in the multienzyme complex (the editosome) that catalyzes editing and of the recombinant proteins were characterized and found to be similar. Ligation of two RNA fragments was enhanced when bridged by a complementary RNA or DNA, which left no gaps or overhangs. An acceptor nucleotide preference of G>U>C>A was observed in the absence of exogenous ATP but U was preferred upon addition of ATP and ligase activity was increased. The substrate specificity and catalytic characteristics indicate that RNA ligase activity contributes to the accuracy of RNA editing.

Animals↗
PubMed2003

Coprinus cinereus DNA ligase I during meiotic development.

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.

Amino Acid Sequence↗
PubMed1999

Nucleotide sequence, heterologous expression and novel purification of DNA ligase from Bacillus stearothermophilus(1).

The gene for DNA ligase (EC 6.5.1.2) from thermophilic bacterium Bacillus stearothermophilus NCA1503 has been cloned and the complete nucleotide sequence determined. The ligase gene encodes a protein 670 amino acids in length. The gene was overexpressed in Escherichia coli and the enzyme has been purified to homogeneity. Preliminary characterisation confirms that it is a thermostable, NAD(+)-dependent DNA ligase.

Amino Acid Sequence↗
PubMed1998

The CHO XRCC1 mutant, EM9, deficient in DNA ligase III activity, exhibits hypersensitivity to camptothecin independent of DNA replication.

We have analyzed the X-ray-sensitive CHO mutant cell line EM9 for sensitivity to the topoisomerase I inhibitor comptothecin. These cells exhibit defective repair of single strand DNA breaks. Recently, EM9 were complemented the DNA ligase III interactive protein, XRCC1. Defective XRCC1 apparently accounts for the low DNA ligase III activity that may explain the single-strand break repair deficiency of EM9 cells. Here, we demonstrate cytotoxic hypersensitivity of EM9 cells following a brief camptothecin treatment. Both the S-phase and non-S-phase populations of EM9 exhibited camptothecin sensitivity relative to the parent cell line AA8. In AA8 cells, only the 55% of the population corresponding to the S-phase subpopulation were sensitive to camptothecin, while the remainder of the population were totally resistant to doses as high as 10 microM. The role of DNA replication in the camptothecin sensitivity was studied using the DNA polymerase inhibitor aphidicolin in co-treatment with camptothecin. Aphidicolin treatment fully protected AA8 cells from camptothecin cytotoxicity. In EM9 cells, aphidicolin protected the S-phase fraction to some degree but all the cells remained sensitive to camptothecin cytotoxicity. These results suggest that EM9 cells are sensitized to camptothecin by a mechanism that is independent of DNA replication and may be a consequence of the XRCC1 mutation or the associated deficiency in DNA ligase III activity. Mechanistic models for the replication-independent cytotoxicity of camptothecin in EM9 cells are discussed.

Animals↗
PubMed2000

A putative ubiquitin ligase required for efficient mRNA export differentially affects hnRNP transport.

BACKGROUND: In the nucleus, mRNAs are bound by hnRNP proteins. A subset of hnRNP proteins shuttle between the nucleus and cytoplasm and are believed to promote mRNA export by acting as adaptors between mRNA and the transport machinery. The existence of multiple shuttling hnRNP proteins raises the question of whether differentially regulated, hnRNP-specific mRNA export pathways exist. RESULTS: We have determined that Tom1p, a conserved protein with a hect (homology to E6-AP carboxyl terminus) E3 ubiquitin ligase domain, is required for efficient mRNA export in S. cerevisiae, yet differentially affects hnRNP protein localization and export. Mutations in tom1 predicted to abolish ubiquitin ligase activity block efficient export of Nab2p and mRNA, causing Nab2p-mRNA complexes to accumulate in a punctate pattern coincident with the nuclear pore complex (NPC). Notably, the subcellular distribution of several other hnRNP proteins is not affected. In particular, Np13p remains mRNA-associated and continues to be efficiently exported in tom1 mutants. CONCLUSION: Our results demonstrate that mutations predicted to affect the enzymatic activity of the Tom1p ubiquitin ligase differentially affect export of hnRNP proteins in association with mRNA. We propose the existence of multiple mRNA export pathways, with export of Nab2p-associated mRNAs dependent on a branch of the ubiquitin protein modification pathway.

Amino Acid Sequence↗
PubMed2003

BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases.

Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. We show here that S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. We propose that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes.

Binding Sites↗
PubMed2004

Ozz-E3, a muscle-specific ubiquitin ligase, regulates beta-catenin degradation during myogenesis.

The identities of the ubiquitin-ligases active during myogenesis are largely unknown. Here we describe a RING-type E3 ligase complex specified by the adaptor protein, Ozz, a novel SOCS protein that is developmentally regulated and expressed exclusively in striated muscle. In mice, the absence of Ozz results in overt maturation defects of the sarcomeric apparatus. We identified beta-catenin as one of the target substrates of the Ozz-E3 in vivo. In the differentiating myofibers, Ozz-E3 regulates the levels of sarcolemma-associated beta-catenin by mediating its degradation via the proteasome. Expression of beta-catenin mutants that reduce the binding of Ozz to endogenous beta-catenin leads to Mb-beta-catenin accumulation and myofibrillogenesis defects similar to those observed in Ozz null myocytes. These findings reveal a novel mechanism of regulation of Mb-beta-catenin and the role of this pool of the protein in myofibrillogenesis, and implicate the Ozz-E3 ligase in the process of myofiber differentiation.

Aging↗
PubMed2002

The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants.

There are two general pathways by which multicellular eukaryotes repair double-strand DNA breaks (DSB): homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). All mammalian mutants in the NHEJ pathway demonstrate a lack of B and T lymphocytes and ionizing radiation sensitivity. Among these NHEJ mutants, the DNA-PK(cs) and Artemis mutants are the least severe, having no obvious phenotype other than the general defects described above. Ku mutants have an intermediate severity with accelerated senescence. The XRCC4 and DNA ligase IV mutants are the most severe, resulting in embryonic lethality. Here we show that the lethality of DNA ligase IV-deficiency in the mouse can be rescued when Ku86 is also absent. To explain the fact that simultaneous gene mutations in the NHEJ pathway can lead to viability when a single mutant is not viable, we propose a nuclease/ligase model. In this model, disrupted NHEJ is more severe if the Artemis:DNA-PK(cs) nuclease is present in the absence of a ligase, and Ku mutants are of intermediate severity, because the nuclease is less efficient. This model is also consistent with the order of severity in organismal phenotypes; consistent with chromosomal breakage observations reported here; and consistent with the NHEJ mutation identified in radiation sensitive human SCID patients.

Animals↗
PubMed1987

Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase.

Preparation and analysis of a series of mutants of bacteriophage T4 RNA ligase that carry single amino acid changes at or near the site of covalent reaction with ATP (adenylylation) are described. The mutant proteins were constructed by site-directed mutagenesis of the gene for T4 RNA ligase (g63) cloned in M13 vectors, transfer of the mutant genes into a lambda pL-containing expression plasmid, and subsequent expression in Escherichia coli. The results give further evidence that Lys-99 is the adenylylation site and that the residue is also important to step 3 in the RNA ligase mechanism (ligation between acceptor and adenylylated donor). Mutations at Glu-100 or Asp-101 have no effect on adenylylation, but Asp-101 is shown to be crucial to both step 2 (transfer of adenylyl to donor) and step 3.

Adenosine Triphosphate↗
PubMed1978

Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61--77 from Escherichia coli tRNAfMet.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.

Base Sequence↗
PubMedDate not supplied

DNA ligase I deficiency in Bloom's syndrome.

Certain rare human diseases with autosomal recessive mode of inheritance are associated with a greatly increased cancer frequency which may reflect specific defects in DNA repair or replication. These disorders include xeroderma pigmentosum, ataxia-telangiectasia, Fanconi's anaemia and Bloom's syndrome. Cells from individuals with Bloom's syndrome usually grow slowly in culture and exhibit increased chromosomal breakage and rearrangement, an elevated frequency of sister chromatid exchanges, retarded rates of progression of DNA replication forks, delayed conversion of replication intermediates to high-molecular-weight DNA, and slightly increased sensitivity to DNA-damaging agents. Several of these features are also characteristic of Escherichia coli and yeast mutants with a defective DNA ligase. In this investigation we show that one of the two DNA ligases of human cells, ligase I, is defective in a representative lymphoid cell line of Bloom's syndrome origin.

Bloom Syndrome↗
PubMed2001

TGF-beta induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that targets SnoN for degradation.

The receptor-regulated Smad proteins are essential intracellular mediators of signal transduction by the transforming growth factor-beta (TGF-beta) superfamily of growth factors and are also important as regulators of gene transcription. Here we describe a new role for TGF-beta-regulated Smad2 and Smad3 as components of a ubiquitin ligase complex. We show that in the presence of TGF-beta signalling, Smad2 interacts through its proline-rich PPXY motif with the tryptophan-rich WW domains of Smurf2, a recently identified E3 ubiquitin ligases. TGF-beta also induces the association of Smurf2 with the transcriptional co-repressor SnoN and we show that Smad2 can function to mediate this interaction. This allows Smurf2 HECT domain to target SnoN for ubiquitin-mediated degradation by the proteasome. Thus, stimulation by TGF-beta can induce the assembly of a Smad2-Smurf2 ubiquitin ligase complex that functions to target substrates for degradation.

Amino Acid Motifs↗
PubMed2003

Identification of the ubiquitin-protein ligase that recognizes oxidized IRP2.

The ubiquitin system is involved in several basic cellular functions. Ubiquitination is carried out by a cascade of three reactions catalysed by the E1, E2 and E3 enzymes. Among these, the E3 ubiquitin-protein ligases have a pivotal role in determining the specificity of the system by recognizing the target substrates through defined targeting motifs. Although RING finger proteins constitute an important family of E3 ligases, only a few post-transcriptional modifications, including phosphorylation, proline hydroxylation and glycosylation, are known to function as recognition signals for E3. Iron regulatory protein 2 (IRP2), a modulator of iron metabolism, is regulated by iron-induced ubiquitination and degradation. Here we show that the RING finger protein HOIL-1 functions as an E3 ligase for oxidized IRP2, suggesting that oxidation is a specific recognition signal for ubiquitination. The oxidation of IRP2 is generated by haem, which binds to IRP2 in iron-rich cells, and by oxygen, indicating that the iron sensing of IRP2 depends on the synthesis and availability of haem.

Amino Acid Sequence↗
PubMed2003

Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex.

BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RINGRING heterodimer. The BRCA1BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn(2+)-loops and central alpha-helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.

Amino Acid Sequence↗
PubMed2002

Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain.

Protein degradation by the ubiquitin (Ub) system controls the concentrations of many regulatory proteins. The degradation signals (degrons) of these proteins are recognized by the system's Ub ligases (complexes of E2 and E3 enzymes). Two substrate-binding sites of UBR1, the E3 of the N-end rule pathway in the yeast Saccharomyces cerevisiae, recognize basic (type 1) and bulky hydrophobic (type 2) N-terminal residues of proteins or short peptides. A third substrate-binding site of UBR1 targets CUP9, a transcriptional repressor of the peptide transporter PTR2, through an internal (non-N-terminal) degron of CUP9. Previous work demonstrated that dipeptides with destabilizing N-terminal residues allosterically activate UBR1, leading to accelerated in vivo degradation of CUP9 and the induction of PTR2 expression. Through this positive feedback, S. cerevisiae can sense the presence of extracellular peptides and react by accelerating their uptake. Here, we show that dipeptides with destabilizing N-terminal residues cause dissociation of the C-terminal autoinhibitory domain of UBR1 from its N-terminal region that contains all three substrate-binding sites. This dissociation, which allows the interaction between UBR1 and CUP9, is strongly increased only if both type 1- and type 2-binding sites of UBR1 are occupied by dipeptides. An aspect of autoinhibition characteristic of yeast UBR1 also was observed with mammalian (mouse) UBR1. The discovery of autoinhibition in Ub ligases of the UBR family indicates that this regulatory mechanism may also control the activity of other Ub ligases.

Amino Acid Sequence↗
PubMed2002

Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu.

Overexpression of the transmembrane receptor tyrosine kinase ErbB2 is common in multiple malignancies, including breast and ovarian cancer. ErbB2 is resistant to degradation mediated by c-Cbl, the E3 ubiquitin ligase responsible for ligand-induced ubiquitination of ErbB1 (epidermal growth factor receptor). Because of its resistance to degradation, ErbB2 is the preferred dimerization partner for other members of the ErbB family, and its overexpression in vivo is associated with poor prognosis. We now show that the chaperone-binding ubiquitin ligase CHIP efficiently ubiquitinates and down-regulates ErbB2. CHIP expression shortens the half-life of both nascent and mature ErbB2 protein. In vitro ubiquitination assay shows that CHIP serves as a ubiquitin ligase for ErbB2, and both exogenously expressed and endogenous CHIP coprecipitate with the kinase. Furthermore, CHIP association with ErbB2 requires a chaperone intermediate and is increased by the chaperone-binding drug geldanamycin, a potent stimulator of ErbB2 ubiquitination and degradation. These data describe a previously unrecognized pathway, amenable to pharmacologic manipulation, that mediates ErbB2 stability.

Animals↗