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Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.
Ando K, Shah AK, Sachdev V, Kleinstiver BP, Taylor-Parker J, Welch MM, Hu Y, Salgia R, White FM, Parvin JD, Ozonoff A, Rameh LE, Joung JK, Bharti AK
(2017) Oncotarget 8: 43733-43751
MeSH Terms: BRCA1 Protein, Camptothecin, Cell Line, Tumor, DNA Topoisomerases, Type I, DNA-Binding Proteins, Drug Resistance, Neoplasm, Gene Editing, Humans, Ku Autoantigen, Multiprotein Complexes, PTEN Phosphohydrolase, Phosphorylation, Proteasome Endopeptidase Complex, Protein Binding, Protein Kinase C, Proteolysis, RNA Interference, Topoisomerase I Inhibitors, Ubiquitin
Show Abstract · Added November 26, 2018
Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.
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MeSH Terms
Human embryonic stem cells have constitutively active Bax at the Golgi and are primed to undergo rapid apoptosis.
Dumitru R, Gama V, Fagan BM, Bower JJ, Swahari V, Pevny LH, Deshmukh M
(2012) Mol Cell 46: 573-83
MeSH Terms: Acetylation, Antigens, Nuclear, Apoptosis, Biological Transport, DNA Damage, DNA-Binding Proteins, Embryonic Stem Cells, Gene Silencing, Genes, bcl-2, Golgi Apparatus, Humans, Ku Autoantigen, Mitochondria, Tumor Suppressor Protein p53, bcl-2-Associated X Protein
Show Abstract · Added October 26, 2015
Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having preactivated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active, and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development.
Copyright © 2012 Elsevier Inc. All rights reserved.
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15 MeSH Terms
Hdm2 is a ubiquitin ligase of Ku70-Akt promotes cell survival by inhibiting Hdm2-dependent Ku70 destabilization.
Gama V, Gomez JA, Mayo LD, Jackson MW, Danielpour D, Song K, Haas AL, Laughlin MJ, Matsuyama S
(2009) Cell Death Differ 16: 758-69
MeSH Terms: Antigens, Nuclear, Apoptosis, Cell Line, Cell Survival, DNA-Binding Proteins, HeLa Cells, Humans, Ku Autoantigen, Phosphorylation, Proto-Oncogene Proteins c-akt, Proto-Oncogene Proteins c-mdm2, RNA, Small Interfering, Tumor Suppressor Protein p53, Ubiquitination, Vascular Endothelial Growth Factor A, bcl-2-Associated X Protein
Show Abstract · Added October 26, 2015
Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.
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16 MeSH Terms
A C-terminal fragment of Cyclin E, generated by caspase-mediated cleavage, is degraded in the absence of a recognizable phosphodegron.
Plesca D, Mazumder S, Gama V, Matsuyama S, Almasan A
(2008) J Biol Chem 283: 30796-803
MeSH Terms: Antigens, Nuclear, Apoptosis, Boronic Acids, Bortezomib, Caspases, Cell Cycle Proteins, Cell Line, Tumor, Cullin Proteins, Cyclin E, DNA-Binding Proteins, F-Box Proteins, F-Box-WD Repeat-Containing Protein 7, Half-Life, Hematologic Neoplasms, Humans, Isoenzymes, Ku Autoantigen, Neoplasm Proteins, Phosphorylation, Protease Inhibitors, Proteasome Endopeptidase Complex, Proto-Oncogene Proteins c-mdm2, Pyrazines, S-Phase Kinase-Associated Proteins, Ubiquitin-Protein Ligases, Ubiquitination, bcl-2-Associated X Protein
Show Abstract · Added October 26, 2015
We have previously shown that caspase-mediated cleavage of Cyclin E generates p18-Cyclin E in hematopoietic tumor cells. Its expression can induce apoptosis or sensitize to apoptotic stimuli in many cell types. However, p18-cyclin E has a much shorter half-life than Cyclin E, being more effectively ubiquitinated and degraded by the 26 S proteasome. A two-step process has emerged that regulates accelerated degradation of Cyclin E, with a caspase-mediated cleavage followed by enhanced proteasome-mediated degradation. We show that recognition of p18-Cyclin E by the Skp1-Cul1-Fbw7 (SCF) complex and its interaction with the Fbw7 protein isoforms can take place independently of phosphorylation of p18-Cyclin E at a C-terminal phosphodegron. In addition to the SCF(Fbw7) pathway, Ku70 binding that facilitates Hdm2 recruitment may also be implicated in p18-Cyclin E ubiquitination. Blocking p18-Cyclin E degradation with proteasome inhibitors increases levels of p18-Cyclin E and enhances its association with Ku70, thus leading to Bax release, its activation, and apoptosis. Moreover, cells expressing p18-Cyclin E are more sensitive to treatment with proteasome inhibitors, such as Bortezomib. By preventing its proteasomal degradation, p18-Cyclin E, but not Cyclin E, may become an effective therapeutic target for Bortezomib and apoptotic effectors in hematopoietic malignancies.
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27 MeSH Terms
Ku70 is stabilized by increased cellular SUMO.
Yurchenko V, Xue Z, Gama V, Matsuyama S, Sadofsky MJ
(2008) Biochem Biophys Res Commun 366: 263-8
MeSH Terms: Animals, Antigens, Nuclear, CHO Cells, Cricetinae, Cricetulus, DNA-Binding Proteins, Ku Autoantigen, Signal Transduction, Small Ubiquitin-Related Modifier Proteins
Show Abstract · Added October 26, 2015
Ku70 is a protein that finds itself at the heart of several important cellular processes. It is essential to the non-homologous end joining pathway as a part of the DNA-end binding complex, required for proper maintenance of telomeres and contributes to DNA damage recognition and regulation of apoptosis. Forces that regulate Ku70 are therefore likely to have large consequences on the physiologic state of the cell. We report here that transient expression of the small protein SUMO resulted in a dramatic increase in the abundance of Ku70. Surprisingly, the direct SUMOylation of Ku70 does not appear to be required for this effect. Rather, Ku70 appears to be stabilized through indirect effects on the rate of degradation. The same outcome was obtained by raising the expression of enzymes that promote SUMOylation. It is likely that many other proteins will be similarly regulated, providing a general control of cellular state.
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9 MeSH Terms
Bax-inhibiting peptide protects cells from polyglutamine toxicity caused by Ku70 acetylation.
Li Y, Yokota T, Gama V, Yoshida T, Gomez JA, Ishikawa K, Sasaguri H, Cohen HY, Sinclair DA, Mizusawa H, Matsuyama S
(2007) Cell Death Differ 14: 2058-67
MeSH Terms: Acetylation, Animals, Antigens, Nuclear, Caspases, Cell Death, Cell Line, Cyclic AMP Response Element-Binding Protein, Cytoprotection, DNA Fragmentation, DNA-Binding Proteins, Histone Acetyltransferases, Humans, Ku Autoantigen, Mutant Proteins, Peptides, Protein Binding, Protein Conformation, Rats, Rats, Sprague-Dawley, Resveratrol, Stilbenes, Vacuoles, bcl-2-Associated X Protein
Show Abstract · Added October 26, 2015
Polyglutamine (polyQ) diseases, such as Huntington's disease and Machado-Joseph disease (MJD), are caused by gain of toxic function of abnormally expanded polyQ tracts. Here, we show that expanded polyQ of ataxin-3 (Q79C), a gene that causes MJD, stimulates Ku70 acetylation, which in turn dissociates the proapoptotic protein Bax from Ku70, thereby promoting Bax activation and subsequent cell death. The Q79C-induced cell death was significantly blocked by Ku70 or Bax-inhibiting peptides (BIPs) designed from Ku70. Furthermore, expression of SIRT1 deacetylase and the addition of a SIRT1 agonist, resveratrol, reduced Q79C toxicity. In contrast, mimicking acetylation of Ku70 abolished the ability of Ku70 to suppress Q79C toxicity. These results indicate that Bax and Ku70 acetylation play important roles in Q79C-induced cell death, and that BIP may be useful in the development of therapeutics for polyQ diseases.
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23 MeSH Terms
Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides.
Gomez JA, Gama V, Yoshida T, Sun W, Hayes P, Leskov K, Boothman D, Matsuyama S
(2007) Biochem Soc Trans 35: 797-801
MeSH Terms: Animals, Antigens, Nuclear, DNA-Binding Proteins, Drug Delivery Systems, Humans, Ku Autoantigen, Oligopeptides, Protein Sorting Signals, Protein Transport, bcl-2-Associated X Protein
Show Abstract · Added October 26, 2015
We found that Ku70, a known DNA repair factor, has a novel function to bind and inhibit Bax (Bcl-2-associated X protein), a key mediator of apoptosis. Pentapeptides derived from the Bax-binding domain of Ku70 were cell-permeable and protected cells from Bax-mediated apoptosis. These pentapeptides were called BIPs (Bax-inhibiting peptides). BIPs may become a useful therapeutic tool to reduce cellular damage. We also generated BIP mutant pentapeptides that do not inhibit Bax, but retain their cell-penetrating activity. Since both BIPs and BIP mutants are cell-permeable, these peptides were designated CPP5s (cell-penetrating pentapeptides). Among the CPP5s discovered, VPTLK (BIP) and KLPVM (BIP mutant) were confirmed to possess protein transduction activity by examination of the delivery of GFP (green fluorescent protein) into cells by these peptides. The mechanism of cell penetration by CPP5s is not known. CPP5s enter the cell at 0 and 4 degrees C. In preliminary studies, various inhibitors of endocytosis and pinocytosis did not show any significant suppression of CPP5 cell entry. CPP5s have very low toxicity in vitro and in vivo and so may be useful tools in order to develop non-toxic drug-delivery technologies.
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10 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
Bax-inhibiting peptide derived from mouse and rat Ku70.
Yoshida T, Tomioka I, Nagahara T, Holyst T, Sawada M, Hayes P, Gama V, Okuno M, Chen Y, Abe Y, Kanouchi T, Sasada H, Wang D, Yokota T, Sato E, Matsuyama S
(2004) Biochem Biophys Res Commun 321: 961-6
MeSH Terms: Amino Acid Sequence, Animals, Antigens, Nuclear, Apoptosis, Cell Line, Cells, Cultured, DNA-Binding Proteins, Etoposide, Female, Humans, Ku Autoantigen, Mice, Molecular Sequence Data, Oligopeptides, Ovary, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-bcl-2, Rats, Sequence Homology, Amino Acid, bcl-2-Associated X Protein
Show Abstract · Added October 26, 2015
Bax is a proapoptotic protein that plays a key role in the induction of apoptosis. Ku70 has activities to repair DNA damage in the nucleus and to suppress apoptosis by inhibiting Bax in the cytosol. We previously designed peptides based on the amino acid sequence of Bax-binding domain of human Ku70, and showed that these peptides bind Bax and inhibit cell death in human cell lines. In the present report, we examined the biological activities of other pentapeptides, VPTLK and VPALR, derived from mouse and rat Ku70. Cells in culture accumulated FITC-labeled VPTLK and VPALR, indicating that these peptides are cell permeable (human, mouse, rat, and porcine cells were examined). These peptides bound to Bax and suppressed cell death in various cell types including primary cultured cells. These data suggest that such Bax inhibiting peptides from three mammalian species may be used to protect healthy cells from apoptotic injury under pathological conditions.
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20 MeSH Terms
Activation of the DNA-dependent protein kinase by drug-induced and radiation-induced DNA strand breaks.
Mårtensson S, Nygren J, Osheroff N, Hammarsten O
(2003) Radiat Res 160: 291-301
MeSH Terms: Aminoglycosides, Anti-Bacterial Agents, Antibiotics, Antineoplastic, Antigens, Nuclear, Antimetabolites, Antineoplastic, Bleomycin, DNA, DNA Damage, DNA Helicases, DNA Repair, DNA Topoisomerases, Type II, DNA-Activated Protein Kinase, DNA-Binding Proteins, Dose-Response Relationship, Drug, Enediynes, Energy Transfer, Enzyme Activation, Etoposide, Ferritins, Ku Autoantigen, Models, Biological, Nucleic Acid Synthesis Inhibitors, Plasmids, Protein Binding, Protein-Serine-Threonine Kinases, Radiation, Ionizing
Show Abstract · Added March 5, 2014
The DNA-dependent protein kinase (DNA-PK) is a DNA-end activated protein kinase that is required for efficient repair of DNA double-strand breaks (DSBs) and for normal resistance to ionizing radiation. DNA-PK is composed of a DNA-binding subunit, Ku, and a catalytic subunit, DNA-PKcs (PRKDC). We have previously shown that PRKDC is activated when the enzyme interacts with the terminal nucleotides of a DSB. These nucleotides are often damaged when DSBs are introduced by anticancer agents and could therefore prevent recognition by DNA-PK. To determine whether DNA-PK could recognize DNA strand breaks generated by agents used in the treatment of cancer, we damaged plasmid DNA with anticancer drugs and ionizing radiation. The DNA breaks were tested for the ability to activate purified DNA-PK. The data indicate that DSBs produced by bleomycin, calicheamicin and two types of ionizing radiation ((137)Cs gamma rays and N(7+) ions: high and low linear energy transfer, respectively) activate DNA-PK to levels matching the kinase activation obtained with simple restriction endonuclease-induced DSBs. In contrast, the protein-linked DSBs produced by etoposide and topoisomerase II failed to bind and activate DNA-PK. Our findings indicate that DNA-PK recognizes DSBs regardless of chemical complexity but cannot recognize the protein-linked DSBs produced by etoposide and topoisomerase II.
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26 MeSH Terms