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Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer.
Bhuripanyo K, Wang Y, Liu X, Zhou L, Liu R, Duong D, Zhao B, Bi Y, Zhou H, Chen G, Seyfried NT, Chazin WJ, Kiyokawa H, Yin J
(2018) Sci Adv 4: e1701393
MeSH Terms: Amino Acid Sequence, Bacteriophages, Biocatalysis, Cyclin-Dependent Kinase 4, Endoplasmic Reticulum Stress, HEK293 Cells, Humans, Mutant Proteins, Mutation, Peptides, Proteolysis, Reproducibility of Results, Signal Transduction, Substrate Specificity, Tumor Suppressor Protein p53, Tumor Suppressor Proteins, Ubiquitin, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitination
Show Abstract · Added March 24, 2018
E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine -methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.
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20 MeSH Terms
The anaphase promoting complex contributes to the degradation of the S. cerevisiae telomerase recruitment subunit Est1p.
Ferguson JL, Chao WC, Lee E, Friedman KL
(2013) PLoS One 8: e55055
MeSH Terms: Amino Acid Sequence, Anaphase-Promoting Complex-Cyclosome, Animals, Cdh1 Proteins, G1 Phase, Mutation, Protein Stability, Proteolysis, Recombinant Proteins, S Phase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Telomerase, Ubiquitin-Protein Ligase Complexes, Ubiquitination
Show Abstract · Added March 5, 2014
Telomerase is a multi-subunit enzyme that reverse transcribes telomere repeats onto the ends of linear eukaryotic chromosomes and is therefore critical for genome stability. S. cerevisiae telomerase activity is cell-cycle regulated; telomeres are not elongated during G1 phase. Previous work has shown that Est1 protein levels are low during G1 phase, preventing telomerase complex assembly. However, the pathway targeting Est1p for degradation remained uncharacterized. Here, we show that Est1p stability through the cell cycle mirrors that of Clb2p, a known target of the Anaphase Promoting Complex (APC). Indeed, Est1p is stabilized by mutations in both essential and non-essential components of the APC. Mutations of putative Destruction boxes (D-boxes), regions shown to be important for recognition of known APC substrates, stabilize Est1p, suggesting that Est1p is likely to be targeted for degradation directly by the APC. However, we do not detect degradation or ubiquitination of recombinant Est1p by the APC in vitro, suggesting either that the recombinant protein lacks necessary post-translational modification and/or conformation, or that the APC affects Est1p degradation by an indirect mechanism. Together, these studies shed light on the regulation of yeast telomerase assembly and demonstrate a new connection between telomere maintenance and cell cycle regulation pathways.
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15 MeSH Terms
State of the APC/C: organization, function, and structure.
McLean JR, Chaix D, Ohi MD, Gould KL
(2011) Crit Rev Biochem Mol Biol 46: 118-36
MeSH Terms: Anaphase-Promoting Complex-Cyclosome, Animals, Catalysis, Cell Cycle Proteins, Cell Nucleus, Humans, Meiosis, Microscopy, Electron, Mitosis, Models, Biological, Spindle Apparatus, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases
Show Abstract · Added March 5, 2014
The ubiquitin-proteasome protein degradation system is involved in many essential cellular processes including cell cycle regulation, cell differentiation, and the unfolded protein response. The anaphase-promoting complex/cyclosome (APC/C), an evolutionarily conserved E3 ubiquitin ligase, was discovered 15 years ago because of its pivotal role in cyclin degradation and mitotic progression. Since then, we have learned that the APC/C is a very large, complex E3 ligase composed of 13 subunits, yielding a molecular machine of approximately 1 MDa. The intricate regulation of the APC/C is mediated by the Cdc20 family of activators, pseudosubstrate inhibitors, protein kinases and phosphatases and the spindle assembly checkpoint. The large size, complexity, and dynamic nature of the APC/C represent significant obstacles toward high-resolution structural techniques; however, over the last decade, there have been a number of lower resolution APC/C structures determined using single particle electron microscopy. These structures, when combined with data generated from numerous genetic and biochemical studies, have begun to shed light on how APC/C activity is regulated. Here, we discuss the most recent developments in the APC/C field concerning structure, substrate recognition, and catalysis.
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13 MeSH Terms
Structural organization of the anaphase-promoting complex bound to the mitotic activator Slp1.
Ohi MD, Feoktistova A, Ren L, Yip C, Cheng Y, Chen JS, Yoon HJ, Wall JS, Huang Z, Penczek PA, Gould KL, Walz T
(2007) Mol Cell 28: 871-85
MeSH Terms: Anaphase-Promoting Complex-Cyclosome, Cdc20 Proteins, Cell Cycle Proteins, Chromatography, Affinity, Cryoelectron Microscopy, Immunoblotting, Mitosis, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Protein Conformation, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitination
Show Abstract · Added March 5, 2014
The anaphase-promoting complex/cyclosome (APC/C) is a conserved multisubunit E3 ubiquitin (Ub) ligase required to signal the degradation of key cell-cycle regulators. Using single particle cryo-electron microscopy (cryo-EM), we have determined a three-dimensional (3D) structure of the core APC/C from Schizosaccharomyces pombe bound to the APC/C activator Slp1/Cdc20. At the 27 A resolution of our density map, the APC/C is a triangular-shaped structure, approximately 19x17x15 nm in size, with a deep internal cavity and a prominent horn-like protrusion emanating from a lip of the cavity. Using antibody labeling and mutant analysis, we have localized 12 of 13 core APC/C components, as well as the position of the activator Slp1, enabling us to propose a structural model of APC/C organization. Comparison of the APC/C with another multiprotein E3 ligase, the SCF complex, uncovers remarkable structural similarities.
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16 MeSH Terms
SCFCdc4-mediated degradation of the Hac1p transcription factor regulates the unfolded protein response in Saccharomyces cerevisiae.
Pal B, Chan NC, Helfenbaum L, Tan K, Tansey WP, Gething MJ
(2007) Mol Biol Cell 18: 426-40
MeSH Terms: Amino Acid Motifs, Amino Acid Sequence, Anaphase-Promoting Complex-Cyclosome, Basic-Leucine Zipper Transcription Factors, Binding Sites, Cell Cycle Proteins, Cell Nucleus, Cell Survival, Cyclin-Dependent Kinase 8, Cyclin-Dependent Kinases, F-Box Proteins, Mitogen-Activated Protein Kinase Kinases, Molecular Sequence Data, Mutation, Proteasome Endopeptidase Complex, Protein Folding, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Serine, Transcription Factors, Two-Hybrid System Techniques, Ubiquitin, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases
Show Abstract · Added March 10, 2014
The Saccharomyces cerevisiae basic leucine zipper transcription factor Hac1p is synthesized in response to the accumulation of unfolded polypeptides in the lumen of the endoplasmic reticulum (ER), and it is responsible for up-regulation of approximately 5% of all yeast genes, including ER-resident chaperones and protein-folding catalysts. Hac1p is one of the most short-lived yeast proteins, having a half-life of approximately 1.5 min. Here, we have shown that Hac1p harbors a functional PEST degron and that degradation of Hac1p by the proteasome involves the E2 ubiquitin-conjugating enzyme Ubc3/Cdc34p and the SCF(Cdc4) E3 complex. Consistent with the known nuclear localization of Cdc4p, rapid degradation of Hac1p requires the presence of a functional nuclear localization sequence, which we demonstrated to involve basic residues in the sequence (29)RKRAKTK(35). Two-hybrid analysis demonstrated that the PEST-dependent interaction of Hac1p with Cdc4p requires Ser146 and Ser149. Turnover of Hac1p may be dependent on transcription because it is inhibited in cell mutants lacking Srb10 kinase, a component of the SRB/mediator module of the RNA polymerase II holoenzyme. Stabilization of Hac1p by point mutation or deletion, or as the consequence of defects in components of the degradation pathway, results in increased unfolded protein response element-dependent transcription and improved cell viability under ER stress conditions.
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26 MeSH Terms
Role of Hcn1 and its phosphorylation in fission yeast anaphase-promoting complex/cyclosome function.
Yoon HJ, Feoktistova A, Chen JS, Jennings JL, Link AJ, Gould KL
(2006) J Biol Chem 281: 32284-93
MeSH Terms: Alanine, Amino Acid Substitution, Anaphase-Promoting Complex-Cyclosome, Cell Cycle Proteins, Fungal Proteins, G2 Phase, Gene Deletion, Green Fluorescent Proteins, Phosphorylation, Repressor Proteins, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Ubiquitin-Protein Ligase Complexes
Show Abstract · Added April 18, 2013
The anaphase-promoting complex/cyclosome (APC/C) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. The APC/C becomes active at the metaphase/anaphase transition and remains active during G(1) phase. One mechanism linked to activation of the APC/C is phosphorylation. Although many sites of mitotic phosphorylation have been identified in core components of the APC/C, the consequence of any individual phosphorylation event has not been elucidated in vivo. In this study, we show that Hcn1 is an essential core component of the fission yeast APC/C and is critical for maintaining complex integrity. Moreover, Hcn1 is a phosphoprotein in vivo. Phosphorylation of Hcn1 occurs at a single Cdk1 site in vitro and in vivo. Mutation of this site to alanine, but not aspartic acid, compromises APC/C function and leads to a specific defect in the completion of cell division.
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13 MeSH Terms
Pressure-induced regulation of IL-6 in retinal glial cells: involvement of the ubiquitin/proteasome pathway and NFkappaB.
Sappington RM, Calkins DJ
(2006) Invest Ophthalmol Vis Sci 47: 3860-9
MeSH Terms: Animals, Animals, Newborn, Astrocytes, Cell Separation, Cells, Cultured, Cysteine Proteinase Inhibitors, Enzyme-Linked Immunosorbent Assay, Fluorescent Antibody Technique, Indirect, Hydrostatic Pressure, Interleukin-6, Leupeptins, Microglia, NF-kappa B, RNA, Messenger, Rats, Rats, Sprague-Dawley, Retina, Reverse Transcriptase Polymerase Chain Reaction, Tetradecanoylphorbol Acetate, Ubiquitin-Protein Ligase Complexes
Show Abstract · Added May 27, 2014
PURPOSE - To investigate how hydrostatic pressure influences regulation of interleukin (IL)-6 by retinal glia and whether this regulation is associated with the ubiquitin/proteasome pathway (UPP) and activation of the transcription factor nuclear factor (NF)kappaB.
METHODS - Astrocytes and microglia isolated from rat retina were maintained in vitro, and the IL-6 concentration in the media at ambient and elevated pressure were compared, with and without the proteasome inhibitor MG132 (10 microM). Immunocytochemistry was used to correlate translocation of NFkappaB with pressure.
RESULTS - Exposure to elevated pressure for 24 hours maximally altered the concentration of media IL-6 of glia cultures, where IL-6 concentrations decreased in astrocyte cultures and increased in microglia cultures. These pressure-induced changes in IL-6 were largely insensitive to MG132 in astrocytes, but were largely MG132-sensitive in microglia. Like IL-6 regulation, pressure-induced activation of NFkappaB also differed between the two glial cell types, where nuclear localization of NFkappaB was transient in astrocytes, but sustained in microglia. Elevated pressure also increased MG132-sensitive expression of IL-6 mRNA by microglia.
CONCLUSIONS - Though pressure-induced regulation of IL-6 by astrocytes is preceded by NFkappaB translocation, it is not altered by MG132 and therefore is not likely to be regulated by NFkappaB or the UPP. In contrast, pressure-induced regulation of IL-6 protein and mRNA by microglia is preceded by NFkappaB translocation and is sensitive to MG132. Together with precedence in the literature, these data suggest that pressure-induced activation of the UPP leads to transcription of IL-6 driven by NFkappaB.
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20 MeSH Terms
Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis.
Gama V, Yoshida T, Gomez JA, Basile DP, Mayo LD, Haas AL, Matsuyama S
(2006) Exp Cell Res 312: 488-99
MeSH Terms: Acetylcysteine, Amino Acid Chloromethyl Ketones, Antigens, Nuclear, Apoptosis, Caspase Inhibitors, Cell Line, Cysteine Proteinase Inhibitors, DNA-Binding Proteins, Doxorubicin, Gene Expression, HeLa Cells, Humans, Ku Autoantigen, Leupeptins, Proteasome Endopeptidase Complex, Proteasome Inhibitors, Signal Transduction, Staurosporine, Ubiquitin, Ubiquitin-Protein Ligase Complexes
Show Abstract · Added October 26, 2015
Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.
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20 MeSH Terms
Dim1p is required for efficient splicing and export of mRNA encoding lid1p, a component of the fission yeast anaphase-promoting complex.
Carnahan RH, Feoktistova A, Ren L, Niessen S, Yates JR, Gould KL
(2005) Eukaryot Cell 4: 577-87
MeSH Terms: Anaphase-Promoting Complex-Cyclosome, Apc4 Subunit, Anaphase-Promoting Complex-Cyclosome, Cell Cycle Proteins, Humans, Macromolecular Substances, RNA Splicing, RNA, Messenger, RNA-Binding Proteins, Recombinant Fusion Proteins, Saccharomyces cerevisiae Proteins, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Two-Hybrid System Techniques, Ubiquitin-Protein Ligase Complexes
Show Abstract · Added December 12, 2013
Schizosaccharomyces pombe Dim1p is required for maintaining the steady-state level of the anaphase-promoting complex or cyclosome (APC/C) component Lid1p and thus for maintaining the steady-state level and activity of the APC/C. To gain further insight into Dim1p function, we have investigated the mechanism whereby Dim1p influences Lid1p levels. We show that S. pombe cells lacking Dim1p or Saccharomyces cerevisiae cells lacking its ortholog, Dib1p, are defective in generalized pre-mRNA splicing in vivo, a result consistent with the identification of Dim1p as a component of the purified yeast U4/U6.U5 tri-snRNP complex. Moreover, we find that Dim1p is part of a complex with the splicing factor Prp1p. However, although Dim1p is required for efficient splicing of lid1(+) pre-mRNA, circumventing the necessity for this particular function of Dim1p is insufficient for restoring normal Lid1p levels. Finally, we provide evidence that Dim1p also participates in the nuclear export of lid1(+) mRNA and that it is likely the combined loss of both of these two Dim1p functions which compromises Lid1p levels in the absence of proper Dim1p function. These data indicate that a mechanism acting at the level of mRNA impacts the functioning of the APC/C, a critical complex in controlling mitotic progression.
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14 MeSH Terms
Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast.
Wilkinson CR, Dittmar GA, Ohi MD, Uetz P, Jones N, Finley D
(2004) Curr Biol 14: 2283-8
MeSH Terms: Cell Cycle, Electrophoresis, Polyacrylamide Gel, Flow Cytometry, Genes, Essential, Microscopy, Fluorescence, Mutation, Oligonucleotides, Protein Transport, RNA Splicing, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleoproteins, Small Nuclear, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Two-Hybrid System Techniques, Ubiquitin-Protein Ligase Complexes, Ubiquitins
Show Abstract · Added March 5, 2014
Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well.
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16 MeSH Terms