554 results on '"APC/C"'
Search Results
152. An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast.
- Author
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Horikoshi, Yasunori, Habu, Toshiyuki, and Matsumoto, Tomohiro
- Published
- 2013
- Full Text
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153. Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex.
- Author
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Sedgwick, Garry G, Hayward, Daniel G, Di Fiore, Barbara, Pardo, Mercedes, Yu, Lu, Pines, Jonathon, and Nilsson, Jakob
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UBIQUITIN , *MITOSIS , *PROTEIN kinases , *SPINDLE apparatus , *DIPEPTIDES , *DIMERIZATION , *UBIQUITINATION - Abstract
The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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154. The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells
- Author
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Naoe, Hideaki, Chiyoda, Tatsuyuki, Ishizawa, Jo, Masuda, Kenta, Saya, Hideyuki, and Kuninaka, Shinji
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ANAPHASE , *HEPARIN , *TROPHOBLAST , *CELL cycle , *CYCLINS , *CADHERINS , *PLACENTA , *STEM cells , *FIBROBLAST growth factors - Abstract
Abstract: Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1GT/GT ) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation. [Copyright &y& Elsevier]
- Published
- 2013
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155. The role of Fbxo5 in the development of human malignant tumors.
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Gao J, Yang D, Cao R, Huang H, Ma J, Wang Z, Xia J, and Pan X
- Abstract
Fbxo5 (F-Box only protein 5), as a substrate recognition subunit of SCF (SKP1-Cullin1-Fbox) protein, plays a crucial role in various cellular processes through ubiquitination and degradation of multiple proteins. In recent years, many studies have pointed out that Fbxo5 is critically involved in carcinogenesis. Moreover, targeting Fbxo5 could have a therapeutic potential for cancer therapy. This review focuses on the functions of Fbxo5 in various types of human malignancies and its underlying molecular mechanisms. This review might lay the foundation for enhancing future investigation on Fbxo5 functions in cancer development and progression., Competing Interests: None., (AJCR Copyright © 2022.)
- Published
- 2022
156. Regulation of APC/C-Cdh1 and Its Function in Neuronal Survival.
- Author
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Almeida, Angeles
- Abstract
Neurons are post-mitotic cells that undergo an active downregulation of cell cycle-related proteins to survive. The activity of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells, plays a relevant role in post-mitotic neurons. Recent advances in the study of the regulation of APC/C have documented that the APC/C-activating cofactor, Cdh1, is essential for the function(s) of APC/C in neuronal survival. Here, we review the normal regulation of APC/C activity in proliferating cells and neurons. We conclude that in neurons the APC/C-Cdh1 complex actively downregulates the stability of the cell cycle protein cyclin B1 and the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3. Keeping these proteins destabilized is critical both for preventing the aberrant reentry of post-mitotic neurons into the cell cycle and for maintaining their reduced antioxidant status. Further understanding of the pathophysiological regulation of these proteins by APC/C-Cdh1 in neurons will be important for the search for novel therapeutic targets against neurodegeneration. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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157. Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification.
- Author
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Prosser, Suzanna L., Samant, Mugdha D., Baxter, Joanne E., Morrison, Ciaran G., and Fry, Andrew M.
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CENTROSOMES , *CENTRIOLES , *MITOSIS , *HYDROXYUREA , *DNA damage , *CELL cycle - Abstract
Centrosome duplication is licensed by the disengagement, or 'uncoupling', of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the anaphase-promoting complex (APC/C), leading to securin degradation and release of active separase. Although APC/C activation during G2 arrest is dependent on polo-like kinase 1 (Plk1)-mediated degradation of the APC/C inhibitor, early mitotic inhibitor 1 (Emi1), Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulates centriole disengagement in response to hydroxyurea or DNA damage-induced cell-cycle arrest and this leads to centrosome amplification. However, the reduplication of disengaged centrioles is dependent on cyclin-dependent kinase 2 (Cdk2) activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Although release from these arrests leads to mitotic entry, the presence of disengaged and/or amplified centrosomes results in the formation of abnormal mitotic spindles that lead to chromosome mis-segregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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158. Substrate targeting by the ubiquitin–proteasome system in mitosis
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Min, Mingwei and Lindon, Catherine
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MITOSIS , *UBIQUITIN , *PROTEASOMES , *CYCLIN-dependent kinases , *ANAPHASE , *POST-translational modification , *SPINDLE apparatus - Abstract
Abstract: Both cell cycle progression and the ubiquitin–proteasome system (UPS) that drives it are precisely regulated. Enzymatically, many ubiquitylation and degradation reactions have been characterized in in vitro systems, providing insights into the fundamental mechanisms of the UPS. Biologically, a range of degradation events depending on a ubiquitin ligase called the Anaphase-Promoting Complex (APC/C), have been shown to control mitotic progression through removal of key substrates with extreme temporal precision. However we are only just beginning to understand how the different enzymatic activities of the UPS act collectively – and in cooperation with other cellular factors – for accurate temporal and spatial control of mitotic substrate levels in vivo. [Copyright &y& Elsevier]
- Published
- 2012
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159. Regulation of E2F1 by APC/CCdh1 via K11 linkage-specific ubiquitin chain formation.
- Author
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Budhavarapu, Varija N., White, Erin D., Mahanic, Christina S., Ligong Chen, Fang-Tsyr Lin, and Weei-Chin Lin
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- 2012
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160. APC/CCdh1 controls the proteasome-mediated degradation of E2F3 during cell cycle exit.
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Zhen Ping, Lim, Ratna, Bashir, Tarig, Pagano, Michele, and Guardavaccaro, Daniele
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- 2012
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161. Cell-cycle-regulated expression of STIL controls centriole number in human cells.
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Arquint, Christian, Sonnen, Katharina F., Stierhof, York-Dieter, and Nigg, Erich A.
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HUMAN cell cycle , *CAENORHABDITIS elegans , *MICROSCOPY , *GENOMES , *PROTEINS , *BIOMOLECULES - Abstract
Control of centriole number is crucial for genome stability and ciliogenesis. Here, we characterize the role of human STIL, a protein that displays distant sequence similarity to the centriole duplication factors Ana2 in Drosophila and SAS-5 in Caenorhabditis elegans. Using RNA interference, we show that STIL is required for centriole duplication in human cells. Conversely, overexpression of STIL triggers the near-simultaneous formation of multiple daughter centrioles surrounding each mother, which is highly reminiscent of the phenotype produced by overexpression of the polo-like kinase PLK4 or the spindle assembly abnormal protein 6 homolog (SAS-6). We further show, by fluorescence and immunoelectron microscopy, that STIL is recruited to nascent daughter centrioles at the onset of centriole duplication and degraded, in an APC/CCdc20-Cdh1-dependent manner, upon passage through mitosis. We did not detect a stable complex between STIL and SAS-6, but the two proteins resemble each other with regard to both localization and cell cycle control of expression. Thus, STIL cooperates with SAS-6 and PLK4 in the control of centriole number and represents a key centriole duplication factor in human cells. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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162. Distinct activities of the anaphase-promoting complex/cyclosome (APC/C) in mouse embryonic cells.
- Author
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Yang, Valerie S., Carter, Stephanie A., Yifan Ng, Hyland, Sarah J., Tachibana-Konwalski, Kikuë, Fisher, Rosemary A., Sebire, Neil J., Seckl, Michael J., Pedersen, Roger A., Laskey, Ronald A., and Gonzalez, Michael A.
- Published
- 2012
- Full Text
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163. Structural insights into anaphase-promoting complex function and mechanism.
- Author
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Barford, David
- Abstract
The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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164. Insights into phosphorylation-dependent mechanisms regulating USP1 protein stability during the cell cycle.
- Author
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Cotto-Rios, Xiomaris M., Jones, Mathew J. K., and Huang, Tony T.
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- 2011
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165. The emerging role of APC/CCdh1 in development
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Hu, Dong, Qiao, Xinxian, Wu, George, and Wan, Yong
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UBIQUITIN , *CELL cycle , *CELL differentiation , *CADHERINS , *MITOSIS , *GENOMES , *DNA replication , *BIOCHEMISTRY , *LABORATORY mice - Abstract
Abstract: The function of APC/C (anaphase-promoting complex/cyclosome) was initially implicated with the onset of anaphase during mitosis, where its association with Cdc20 targets securin for destruction, thereby allowing the separation of two duplicated daughter genomes. When combined with Cdh1, APC regulates G1/S transition and DNA replication during cell cycle. Beyond cell cycle control, results from recent biochemical and mouse genetic studies have attracted our attention to the unexpected impact of APC/CCdh1 in cellular differentiation, genomic integrity and pathogenesis of various diseases. This review will aim to summarize current understanding of APC/CCdh1 in regulating crucial events during development. [Copyright &y& Elsevier]
- Published
- 2011
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166. Correction of microtubule–kinetochore attachment errors: Mechanisms and role in tumor suppression
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Ricke, Robin M. and van Deursen, Jan M.
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MICROTUBULES , *PROTEIN structure , *TUMOR suppressor proteins , *MITOSIS , *CHROMOSOMES , *SISTER chromatid exchange , *CANCER cells , *GENOMES - Abstract
Abstract: During mitosis, cells segregate duplicated chromosomes with high fidelity in order to maintain genome stability. Proper attachment of sister kinetochores to spindle microtubules is critical for accurate chromosome segregation and is driven by complex mechanisms that promote the capture of unattached kinetochores and the resolution of erroneously attached kinetochores. Defects in these surveillance systems promote chromosome segregation and aneuploidy and can contribute to neoplastic transformation. Understanding, how, at the molecular level, accurate chromosome segregation is achieved may be crucial for our understanding of how cancer cells develop genome instability. [Copyright &y& Elsevier]
- Published
- 2011
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167. Processive ubiquitin chain formation by the anaphase-promoting complex
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Meyer, Hermann-Josef and Rape, Michael
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UBIQUITIN , *MITOSIS , *CELL division , *CELL cycle regulation , *NUCLEOTIDES , *PROTEINS - Abstract
Abstract: Progression through mitosis requires the sequential ubiquitination of cell cycle regulators by the anaphase-promoting complex, resulting in their proteasomal degradation. Although several mechanisms contribute to APC/C regulation during mitosis, the APC/C is able to discriminate between its many substrates by exploiting differences in the processivity of ubiquitin chain assembly. Here, we discuss how the APC/C achieves processive ubiquitin chain formation to trigger the sequential degradation of cell cycle regulators during mitosis. [Copyright &y& Elsevier]
- Published
- 2011
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168. Chromium induces chromosomal instability, which is partly due to deregulation of BubR1 and Emi1, two APC/C inhibitors.
- Author
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Liyan Hu, Xin Liu, Chervona, Yana, Feikun Yang, Moon-shong Tang, Darzynkiewicz, Zbigniew, and Wei Dai
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- 2011
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169. New Insights into the Genetic Regulation of Homologue Disjunction in Mammalian Oocytes.
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Homer, H.
- Subjects
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OVUM , *MEIOSIS , *ANEUPLOIDY , *MITOSIS , *CELL cycle regulation , *AGE factors in human body composition - Abstract
Mammalian oocytes execute a unique meiotic programme involving 2 arrest stages and an unusually protracted preamble to chromosome segregation during the first meiotic division (meiosis I). How mammalian oocytes successfully navigate their exceptional meiotic journey has long been a question of immense interest. Understanding the minutiae of female mammalian meiosis I is not merely of academic interest as 80-90% of human aneuploidy is the consequence of errors arising at this particular stage of oocyte maturation, a stage with a peculiar vulnerability to aging. Recent evidence indicates that oocytes employ many of the same cast of proteins during meiosis I as somatic cells do during mitosis, often to execute similar tasks, but intriguingly, occasionally delegate them to unexpected and unprecedented roles. This is epitomised by the master cell-cycle regulon, the anaphase-promoting complex or cyclosome (APC/C), acting in concert with a critical APC/C-targeted surveillance mechanism, the spindle assembly checkpoint (SAC). Together, the APC/C and the SAC are among the most influential entities overseeing the fidelity of cell-cycle progression and the precision of chromosome segregation. Here I review the current status of pivotal elements underpinning homologue disjunction in mammalian oocytes including spindle assembly, critical biochemical anaphase-initiating events, APC/C activity and SAC signalling along with contemporary findings relevant to progressive oocyte SAC dysfunction as a model for age-related human aneuploidy. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]
- Published
- 2011
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170. The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27.
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Ziguo Zhang, Kulkarni, Kiran, Hanrahan, Sarah J., Thompson, Andrew J., and Barford, David
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CELL cycle , *UBIQUITIN , *PROTEIN analysis , *SCHIZOSACCHAROMYCES pombe , *OLIGOMERS , *BIOMOLECULES , *PHYSIOLOGY - Abstract
The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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171. APC/C-Cdh1.
- Author
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Xinxian Qiao, Liyong Zhang, Gamper, Armin M., Fujita, Takeo, and Yong Wan
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- 2010
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172. APC/CCdc20 targets E2F1 for degradation in prometaphase.
- Author
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Peart, Melissa J., Poyurovsky, Masha V., Kass, Elizabeth M., Urist, Marshall, Verschuren, Emmy W., Summers, Matthew K., Jackson, Peter K., and Prives, Carol
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- 2010
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173. Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint
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Holmfeldt, Per, Sellin, Mikael E., and Gullberg, Martin
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CHROMOSOME abnormalities , *MICROTUBULES , *SPINDLE apparatus , *MITOSIS regulation , *TUBULINS , *GENETIC mutation , *TUMOR proteins , *ANEUPLOIDY - Abstract
Abstract: Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18→E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
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174. p53- and p21-dependent premature APC/C–Cdh1 activation in G2 is part of the long-term response to genotoxic stress.
- Author
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Wiebusch, L. and Hagemeier, C.
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DNA damage , *TUMOR suppressor proteins , *GENETIC toxicology , *DNA repair , *AGING - Abstract
The long-term cellular response to DNA damage is controlled by the tumor suppressor p53. It results in cell-cycle arrest followed by DNA repair and, depending on the degree of damage inflicted, premature senescence or apoptotic cell death. Here we show that in normal diploid fibroblasts the ubiquitin ligase anaphase-promoting complex or cyclosome (APC/C)–Cdh1 becomes prematurely activated in G2 as part of the sustained long-term but not the rapid short-term response to genotoxic stress and results in the degradation of numerous APC/C substrates. Using HCT116 somatic knockout cells we show that mechanistically premature APC/C activation depends on p53 and its transcriptional target p21 that mediates the signal through downregulation of the APC/C inhibitor Emi1. Cdc14B is dispensable in this setting but might function redundantly. Our data suggest an unexpected role for the APC/C in executing a part of the p53-dependent DNA damage response that leads to premature senescence. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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175. Molecular Structure of the N-terminal Domain of the APC/C Subunit Cdc27 Reveals a Homo-dimeric Tetratricopeptide Repeat Architecture
- Author
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Zhang, Ziguo, Roe, S. Mark, Diogon, Marie, Kong, Eric, El Alaoui, Hicham, and Barford, David
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MOLECULAR structure , *PEPTIDES , *UBIQUITIN , *LIGASES , *GENE targeting , *DIMERS , *PROTEINS , *CELL cycle - Abstract
Abstract: The anaphase promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that targets specific cell cycle regulatory proteins for ubiquitin-dependent degradation, thereby controlling cell cycle events such as the metaphase to anaphase transition and the exit from mitosis. Biochemical and genetic studies are consistent with the notion that subunits of APC/C are organised into two distinct sub-complexes; a catalytic sub-complex including the cullin domain and RING finger subunits Apc2 and Apc11, respectively, and a tetratricopeptide repeat (TPR) sub-complex composed of the TPR subunits Cdc16, Cdc23 and Cdc27 (Apc3). Here, we describe the crystal structure of the N-terminal domain of Encephalitozoon cuniculi Cdc27 (Cdc27Nterm), revealing a homo-dimeric structure, composed predominantly of successive TPR motifs. Mutation of the Cdc27Nterm dimer interface destabilises the protein, disrupts dimerisation in solution, and abolishes the capacity of E. cuniculi Cdc27 to complement Saccharomyces cerevisiae Cdc27 in vivo. These results establish the existence of functional APC/C genes in E. cuniculi, the evolutionarily conserved dimeric properties of Cdc27, and provide a framework for understanding the architecture of full-length Cdc27. [Copyright &y& Elsevier]
- Published
- 2010
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176. A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes
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Mazanek, Michael, Roitinger, Elisabeth, Hudecz, Otto, Hutchins, James R.A., Hegemann, Björn, Mitulović, Goran, Taus, Thomas, Stingl, Christoph, Peters, Jan-Michael, and Mechtler, Karl
- Subjects
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PEPTIDES , *PHOSPHORYLATION , *TITANIUM dioxide , *ZIRCONIUM oxide , *PHASE partition , *MASS spectrometry , *PROTEINS , *ACETIC acid , *CHROMATOGRAPHIC analysis - Abstract
Abstract: The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004 ; Larsen et al., 2005 ; Kweon and Hakansson, 2006 ; Cantin et al., 2007 ; Collins et al., 2007 ). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO2-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007 ). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO2 and ZrO2 columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes. [Copyright &y& Elsevier]
- Published
- 2010
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177. Anaphase-promoting complex/cyclosome-cdh1 mediates the ubiquitination and degradation of TRB3
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Ohoka, Nobumichi, Sakai, Satoshi, Onozaki, Kikuo, Nakanishi, Makoto, and Hayashi, Hidetoshi
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PROTEINS , *MITOSIS , *UBIQUITIN , *ENDOPLASMIC reticulum , *CELL death , *CELLULAR control mechanisms , *PROTEOLYSIS , *AMINO acids - Abstract
Abstract: We have recently demonstrated that TRB3, a novel endoplasmic reticulum (ER) stress-inducible protein, is induced by CHOP and ATF4 to regulate their function and ER stress-induced cell death; however, the regulation of TRB3 function has not been well characterized. Here we demonstrate that TRB3 is an unstable protein regulated by the ubiquitin–proteasome system. The carboxyl-terminal domain of TRB3 is necessary for protein degradation, and in this region, we found the typical D-box motif, which is a critical sequence for the anaphase-promoting complex/cyclosome (APC/C) dependent proteolysis. TRB3 proteins were stabilized by deletion of its D-box motif and interacted with APC/C coactivator proteins, Cdc20 and Cdh1. The expression level of TRB3 protein is down-regulated by over-expression of Cdh1 but not by that of Cdc20. In addition, knockdown of Cdh1 enhanced the endogenous TRB3 expression level and suppressed its ubiquitination level. These results suggest that APC/CCdh1 is involved in ubiquitination and down-regulating the stability of TRB3 protein. [Copyright &y& Elsevier]
- Published
- 2010
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178. On the role of the chaperonin CCT in the just-in-time assembly process of APC/CCdc20
- Author
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Dekker, Carien
- Subjects
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MOLECULAR chaperones , *TRANSCRIPTION factors , *CELL division , *GENETIC regulation , *EUKARYOTIC cells , *CELL cycle - Abstract
Abstract: The just-in-time hypothesis relates to the assembly of large multi-protein complexes and their regulation of activation in the cell. Here I postulate that chaperonins may contribute to the timely assembly and activation of such complexes. For the case of anaphase promoting complex/cyclosomeCdc20 assembly by the eukaryotic chaperonin chaperonin containing Tcp1 it is shown that just-in-time synthesis and chaperone-assisted folding can synergise to generate a highly regulated assembly process of a protein complex that is vital for cell cycle progression. Once dependency has been established transcriptional regulation and chaperonin-dependency may have co-evolved to safeguard the timely activation of important multi-protein complexes. [Copyright &y& Elsevier]
- Published
- 2010
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179. Ubiquitin ligase complexes: from substrate selectivity to conjugational specificity.
- Author
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Nagy, Vanja and Dikic, Ivan
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UBIQUITIN , *LIGASES , *MULTIENZYME complexes , *BIOCONJUGATES , *POST-translational modification , *AMMONIUM perchlorate , *ENZYME kinetics - Abstract
Localization, activity and lifespan of a protein are signaled by a small, 8 kDa protein, ubiquitin (Ub). Ub conjugation is a post-translational modification orchestrated by the sequential action of activating (E1), conjugating (E2), and ligating (E3) enzymes. Although a simple combination of an E2 and an E3 enzyme can be sufficient for an active complex, in other cases ubiquitination can occur in the context of large multimeric complexes with enhanced molecular abilities. Here, we review several Ub ligase complexes to highlight strategies governing conjugational specificity, the gained adaptability in substrate specificity, and modulatory flexibility encoded in regulatory components of these diverse multimers. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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180. The pentose-phosphate pathway in neuronal survival against nitrosative stress.
- Author
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Bolaños, Juan P. and Almeida, Angeles
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PENTOSES , *GLUTATHIONE , *NERVOUS system , *GLYCOLYSIS , *PATHOLOGY - Abstract
Neurons are thought to be particularly vulnerable cells against reactive oxygen and nitrogen species (RONS) damage (nitrosative stress), due in part to their weak antioxidant defense and low ability to compensate energy homeostasis. Intriguingly, nitrosative stress efficiently stimulates the rate of the antioxidant pentose-phosphate pathway (PPP), which generates NADPH a necessary cofactor for the reduction of glutathione disulfide. In fact, inhibition of PPP sensitizes cultured neurons to glutathione oxidation and apoptotic death, whereas its stimulation confers resistance to nitrosative stress. Furthermore, we recently described that neurons can preferentially use glucose through the PPP by inhibiting glycolysis, which is achieved by continuously degrading the glycolytic positive-effector protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (Pfkfb3) by the action of the E3 ubiquitine ligase anaphase-promoting complex/cyclosome (APC/C)Cdh1. These results suggest that the antioxidant fragility of neurons may be compensated by the PPP at the expense of inhibiting bioenergetic glycolysis. © 2009 IUBMB IUBMB Life, 62(1):14–18, 2010 [ABSTRACT FROM AUTHOR]
- Published
- 2010
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181. BubR1 acetylation at prometaphase is required for modulating APC/C activity and timing of mitosis.
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Eunhee Choi, Hyerim Choe, Jaewon Min, Ji Yoon Choi, Jimi Kim, and Hyunsook Lee
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ACETYLATION , *MITOSIS , *AMINO acids , *UBIQUITIN , *GENE expression - Abstract
Regulation of BubR1 is central to the control of APC/C activity. We have found that BubR1 forms a complex with PCAF and is acetylated at lysine 250. Using mass spectrometry and acetylated BubR1-specific antibodies, we have confirmed that BubR1 acetylation occurs at prometaphase. Importantly, BubR1 acetylation was required for checkpoint function, through the inhibition of ubiquitin-dependent BubR1 degradation. BubR1 degradation began before the onset of anaphase. It was noted that the pre-anaphase degradation was regulated by BubR1 acetylation. Degradation of an acetylation-mimetic form, BubR1–K250Q, was inhibited and chromosome segregation in cells expressing BubR1–K250Q was markedly delayed. By contrast, the acetylation-deficient mutant, BubR1–K250R, was unstable, and mitosis was accelerated in BubR1–K250R-expressing cells. Furthermore, we found that APC/C–Cdc20 was responsible for BubR1 degradation during mitosis. On the basis of our collective results, we propose that the acetylation status of BubR1 is a molecular switch that converts BubR1 from an inhibitor to a substrate of the APC/C complex, thus providing an efficient way to modulate APC/C activity and mitotic timing. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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182. Regulation of Cyclin A protein in meiosis and early embryogenesis.
- Author
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Vardy, Leah, Pesin, Jillian A., and Orr-Weaver, Terry L.
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PROTEINS , *MEIOSIS , *EMBRYOLOGY , *MESSENGER RNA , *OOGENESIS - Abstract
In contrast to the extensive analysis of the regulation of Cyclin B protein levels during developmental progression through meiosis in oogenesis, little is known about Cyclin A. Repression of cyclin A translation early in prophase I in Drosophila is important to maintain the oocyte in meiosis, and this has been shown to be mediated by deadenylation of the mRNA and inhibition by the Bruno repressor. We find that at oocyte maturation as meiosis resumes, Cyclin A protein reappears, coincident with polyadenylation of the mRNA and loss of Bruno repressor. Cyclin A is multiphosphorylated in a pattern consistent with autophosphorylation. and this form accumulates aberrantly in metaphase I if the Cortex form of the Anaphase Promoting Complex/Cyclosome is inactive. The PAN GU (PNG) kinase positively promotes translation of Cyclin A. beginning in oogenesis, an earlier onset than previously recognized. After egg activation and the completion of melosis, PNG promotes further polyadenylation of cyclin A mRNA and appears to antagonize repression of translation by the PUMILIO inhibitor. Epistasis studies with png; apc mutants indicate that PNG acts solely to promote translation, rather than having a parallel function to inhibit degradation. These studies reveal multiple levels of posttranscriptional regulation of Cyclin A protein by translational and proteolytic control during oocyte maturation and the onset of embryogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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183. APC/C CDH1 ubiquitinates IDH2 contributing to ROS increase in mitosis.
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Lambhate, Surbhi, Bhattacharjee, Debanjan, and Jain, Nishant
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LABORATORY mice , *REACTIVE oxygen species , *CELL cycle , *MITOSIS - Abstract
NADPH is a cofactor used by reactive oxygen species (ROS) scavenging enzymes to block ROS produced in cells. Recently, it was shown that in cancer cells, ROS progressively increases in tune to cell cycle leading to a peak in mitosis. Loss of IDH2 is known to cause severe oxidative stress in cell and mouse models as ROS increases in mitochondria. Therefore, we hypothesized that IDH2, a major NADPH-producing enzyme in mitochondria is ubiquitinated for ROS to increase in mitosis. To test this hypothesis, in cancer cells we examined IDH2 ubiquitination in mitosis and measured the ROS produced. We found that IDH2 is ubiquitinated in mitosis and on inhibiting anaphase-promoting complex/Cyclosome (APC/C) IDH2 was stabilized. Further, we observed that overexpressing APC/C coactivator CDH1 decreased IDH2, whereas depleting CDH1 decreased IDH2 ubiquitination. To understand the link between IDH2 ubiquitination and ROS produced in mitosis, we show that overexpressing mitochondria-targeted-IDH1 decreased ROS by increasing NADPH in IDH2 ubiquitinated cells. We conclude that APC/C CDH1 ubiquitinates IDH2, a major NADPH-producing enzyme in mitochondria contributing to ROS increase in mitosis. Based on our results, we suggest that mitosis can be a therapeutic window in mutant IDH2-linked pathologies. Proposed mechanism to show Mito-IDH1 and ubiquitinated IDH2 regulating NADPH and ROS levels in mitosis. [Display omitted] • Wild-type and pathogenic mutants of IDH2 are ubiquitinated by APC/C CDH1 in mitosis. • Ubiquitination of IDH2 contributes to ROS increase in mitosis. • Mito-IDH1 offsets ubiquitinated IDH2. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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184. To cell cycle, swing the APC/C
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van Leuken, Renske, Clijsters, Linda, and Wolthuis, Rob
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CELL cycle , *MITOSIS , *PROTEIN kinases , *CYCLINS - Abstract
Abstract: For successful mitosis, Cyclin B1 and Securin must be degraded efficiently before anaphase. Destruction of these mitotic regulators by the 26S proteasome is the result of their poly-ubiquitination by a multi-subunit E3 ligase: the Anaphase-Promoting Complex or Cyclosome (APC/C). Clearly, the APC/C is not just important for mitosis. Destruction of APC/C substrates such as Cdc20, Plk1, Aurora A and Skp2 directs events in G1. Strikingly, the APC/C needs to stay active even in quiescent cells to keep them out of the cell cycle and forms an intriguing link with pRb. An inactive APC/C stabilizes Geminin, Cyclin A and Cyclin B1, thereby securing completion of DNA synthesis and progression through G2-phase. In prometaphase the APC/C becomes active again, but is controlled by the spindle assembly checkpoint. Here we discuss how the APC/C is either held in check or released. We argue that shedding more light on the APC/C is also important to understand cancer and could help the design of treatment. [Copyright &y& Elsevier]
- Published
- 2008
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185. Two components of the Myb complex, DMyb and Mip130, are specifically associated with euchromatin and degraded during prometaphase throughout development
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Scaria, George S., Ramsay, Gary, and Katzen, Alisa L.
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CELL proliferation , *CELL cycle , *CELL division , *POLYPLOIDY - Abstract
Abstract: The Drosophila Myb protein, DMyb, is a transcription factor important for cell proliferation and development. Unlike the mRNAs produced by mammalian myb genes, Drosophila myb transcripts do not fluctuate substantially during the cell cycle. A comprehensive analysis of the localization and degradation of the DMyb protein has now revealed that DMyb is present in nuclei during S phase of all mitotically active tissues throughout embryogenesis and larval development. However, DMyb and Mip130, another member of the Myb complex, are not uniformly distributed throughout the nucleus. Instead, both proteins, which colocalize, appear to be specifically excluded from heterochromatic regions of chromosomes. Furthermore, DMyb and Mip130 are unstable proteins that are degraded during prometaphase of mitosis. The timing of their degradation is reminiscent of Cyclin A, but at least for DMyb, the mechanism differs; although DMyb degradation is dependent on core APC/C components, it does not depend on the Fizzy or Fizzy-related adaptor proteins. DMyb levels are also high in actively endoreplicating polyploid cells, but there is no indication of cyclical degradation. We conclude that cell cycle specific degradation of DMyb and Mip130 is likely to be utilized as a key regulatory mechanism in down-regulating their levels and the activity of the Myb complex. [Copyright &y& Elsevier]
- Published
- 2008
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186. Mitotic CDKs control the metaphase-anaphase transition and trigger spindle elongation.
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Rahal, Rami and Amon, Angelika
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CELL division , *CELL proliferation , *MITOSIS , *CELL cycle , *CYCLINS , *GROWTH factors , *PROTEIN kinases - Abstract
Mitotic cyclin-dependent kinases (CDKs) control entry into mitosis, but their role during mitotic progression is less well understood. Here we characterize the functions of CDK activity associated with the mitotic cyclins Clb1, Clb2, and Clb3. We show that Clb-CDKs are important for the activation of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C)-Cdc20 that triggers the metaphase-anaphase transition. Furthermore, we define an essential role for Clb-CDK activity in anaphase spindle elongation. Thus, mitotic CDKs serve not only to initiate M phase, but are also needed continuously throughout mitosis to trigger key mitotic events such as APC/C activation and anaphase spindle elongation. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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187. Cdc45 degradation during differentiation and apoptosis
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Pollok, S. and Grosse, F.
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CELL division , *CELL differentiation , *DNA replication , *BIOCHEMISTRY - Abstract
Abstract: Cell division cycle protein 45 (Cdc45) is crucial for the initiation as well as the elongation process of eukaryotic DNA replication. Our findings suggested that the Cdc45 protein is ubiquitylated and degraded by the proteasome pathway in human cells. Firstly, the fate of Cdc45 protein after induction of terminal differentiation of cultured human cells was significantly decelerated by application of proteasomal inhibitors. Secondly, we identified various putative destruction boxes and one KEN-box in the amino acid sequence of vertebrate Cdc45, which indicate that Cdc45 seems to be a novel substrate of the anaphase promoting complex/cyclosome. Thus, the evidences for ubiquitylation of Cdc45 refer the first posttranslational modification of this essential replication factor. We also found, that the human Cdc45 protein was not cleaved during apoptosis of cultured cells. This is in accordance with reports demonstrating the absence of indiscriminative cleavage of replication proteins during the programmed cell death. [Copyright &y& Elsevier]
- Published
- 2007
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188. NuSAP is degraded by APC/C–Cdh1 and its overexpression results in mitotic arrest dependent of its microtubules' affinity
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Li, Lu, Zhou, Ying, Sun, Libo, Xing, Guichun, Tian, Chunyan, Sun, Jing, Zhang, Lingqiang, and He, Fuchu
- Subjects
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TUBULINS , *CELL cycle , *LIGASES , *MICROTUBULES - Abstract
Abstract: Microtubule associated proteins are involved in regulation of microtubule dynamics. Its mutation and dysregulation result in severe consequences such as mitotic block and apoptosis. NuSAP has been reported as a microtubule associated protein, depletion of which by RNAi results in spindle deficiency and cytokinesis failure. However, its role in regulation of cell cycle and how NuSAP protein is controlled during cell cycle progression still remains unclear. Here we show that NuSAP can be ubiquitinated and degraded by APC/C–hCdh1 E3 ligase. Evolutionally conserved KEN box functions as the degron of NuSAP. Overexpression of NuSAP induces mitotic arrest and the microtubule associated domain and nuclear localization are both required for NuSAP to induce mitotic arrest. Furthermore, overexpression of NuSAP results in cells accumulation with microtubule bundling and spindle deficiency. Thus, our results give evidence for the first time that NuSAP protein level is tightly regulated by the APC/C ubiquitin ligase complex and NuSAP induces mitotic arrest dependent of its microtubule affinity. [Copyright &y& Elsevier]
- Published
- 2007
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189. Mutation of the Apc1 homologue shattered disrupts normal eye development by disrupting G1 cell cycle arrest and progression through mitosis
- Author
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Tanaka-Matakatsu, Miho, Thomas, Barbara J., and Du, Wei
- Subjects
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CELL division , *EYE diseases , *KARYOKINESIS , *DROSOPHILA - Abstract
Abstract: The shattered 1 (shtd 1 ) mutation disrupts Drosophila compound eye structure. In this report, we show that the shtd 1 eye defects are due to a failure to establish and maintain G1 arrest in the morphogenetic furrow (MF) and a defect in progression through mitosis. The observed cell cycle defects were correlated with an accumulation of cyclin A (CycA) and String (Stg) proteins near the MF. Interestingly, the failure to maintain G1 arrest in the MF led to the specification of R8 photoreceptor cells that undergo mitosis, generating R8 doublets in shtd 1 mutant eye discs. We demonstrate that shtd encodes Apc1, the largest subunit of the anaphase-promoting complex/cyclosome (APC/C). Furthermore, we show that reducing the dosage of either CycA or stg suppressed the shtd 1 phenotype. While reducing the dosage of CycA is more effective in suppressing the premature S phase entry in the MF, reducing the dosage of stg is more effective in suppressing the progression through mitosis defect. These results indicate the importance of not only G1 arrest in the MF but also appropriate progression through mitosis for normal eye development during photoreceptor differentiation. [Copyright &y& Elsevier]
- Published
- 2007
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190. Mitotic regulation of the anaphase-promoting complex.
- Author
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Baker, D. J., Dawlaty, M. M., Galardy, P., and Van Deursen, J. M.
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- *
MITOSIS , *MICROTUBULES , *CELL cycle , *CELL division , *UBIQUITIN - Abstract
Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C), a large multiprotein E3 ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome. APC/C has two activating subunits, Cdc20 and Cdh1. The well-established view is that Cdc20 activates APC/C from the onset of mitosis through the metaphase-anaphase transition, and that Cdh1 does so from anaphase through G1. Recent work, however, indicates that Cdh1 also activates APC/C in early mitosis and that this APC/C pool targets the anaphase inhibitor securin. To prevent premature degradation of securin, the nuclear transport factors Nup98 and Rae1 associate with APC/CCdh1-securin complexes. In late metaphase, when all kinetochores are attached to spindle microtubules and the spindle assembly checkpoint is satisfied, Nup98 and Rae1 are released from these complexes, thereby allowing for prompt ubiquitination of securin by APC/CCdh1. This, and other mechanisms by which the catalytic activity of APC/C is tightly regulated to ensure proper timing of degradation of each of its mitotic substrates, are highlighted. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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191. Erp1/Emi2 is essential for the meiosis I to meiosis II transition in Xenopus oocytes
- Author
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Ohe, Munemichi, Inoue, Daigo, Kanemori, Yoshinori, and Sagata, Noriyuki
- Subjects
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KARYOKINESIS , *CELL division , *PROTEINS , *VERTEBRATES - Abstract
Abstract: Erp1 (also called Emi2), an inhibitor of the APC/C ubiquitin ligase, is a key component of cytostatic factor (CSF) responsible for Meta-II arrest in vertebrate eggs. Reportedly, however, Erp1 is expressed even during meiosis I in Xenopus oocytes. If so, it is a puzzle why normally maturing oocytes cannot arrest at Meta-I. Here, we show that actually Erp1 synthesis begins only around the end of meiosis I in Xenopus oocytes, and that specific inhibition of Erp1 synthesis by morpholino oligos prevents entry into meiosis II. Furthermore, we demonstrate that premature, ectopic expression of Erp1 at physiological Meta-II levels can arrest maturing oocytes at Meta-I. Thus, our results show the essential role for Erp1 in the meiosis I/meiosis II transition in Xenopus oocytes and can explain why normally maturing oocytes cannot arrest at Meta-I. [Copyright &y& Elsevier]
- Published
- 2007
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192. Autophagy prevents runaway meiotic divisions.
- Author
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Wang, Fei, Denic, Vladimir, and Lacefield, Soni
- Abstract
In budding yeast, macroautophagy/autophagy is required for cells to enter into the meiotic divisions. Our recent publication showed that autophagy is also required for meiotic exit. Inhibition of autophagy as cells enter into the meiotic divisions results in additional rounds of spindle formation, spindle elongation, and aberrant chromosome segregation leading to cell death. Under these conditions, the meiosis II-specific cyclin Clb3 is absent, and two substrates of the anaphase-promoting complex/cyclosome (APC/C) persist into the additional divisions instead of being degraded after meiosis II. We found that the translational repressor Rim4 is a substrate of autophagy, which could explain these observations through its known role in repressing synthesis of Clb3 and the meiosis-specific co-activator of the APC/C, Ama1. Combined, these results provide new mechanistic insight into the control of meiotic exit through timed autophagic degradation of a master regulator of gene expression. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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193. Emi2 Is Essential for Mouse Spermatogenesis
- Author
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Ernesto Guccione, Philipp Kaldis, Vincenzo Coppola, Radoslaw Szmyd, Diana Low, Kui Liu, Ans M.M. van Pelt, Lino Tessarollo, M. Kasim Diril, Heng Yu Chang, Lakshmi Gopinathan, Amsterdam Reproduction & Development (AR&D), and Center for Reproductive Medicine
- Subjects
0301 basic medicine ,Male ,Cdk1 ,Emi2 ,Biology ,F-box protein ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Meiotic Prophase I ,Mice ,0302 clinical medicine ,Meiosis ,spermatocytes ,Animals ,APC/C ,meiosis ,lcsh:QH301-705.5 ,Genetics ,Mice, Knockout ,Cyclin-dependent kinase 1 ,phosphorylation ,F-Box Proteins ,Embryogenesis ,Sperm ,Spermatids ,spermatogenesis ,Cell biology ,diplotene ,030104 developmental biology ,Gene Expression Regulation ,lcsh:Biology (General) ,Knockout mouse ,biology.protein ,Female ,ovary ,Spermatogenesis ,030217 neurology & neurosurgery ,knockout mice - Abstract
The meiotic functions of Emi2, an inhibitor of the APC/C complex, have been best characterized in oocytes where it mediates metaphase II arrest as a component of the cytostatic factor. We generated knockout mice to determine the in vivo functions of Emi2-in particular, its functions in the testis, where Emi2 is expressed at high levels. Male and female Emi2 knockout mice are viable but sterile, indicating that Emi2 is essential for meiosis but dispensable for embryonic development and mitotic cell divisions. We found that, besides regulating cell-cycle arrest in mouse eggs, Emi2 is essential for meiosis I progression in spermatocytes. In the absence of Emi2, spermatocytes arrest in early diplotene of prophase I. This arrest is associated with decreased Cdk1 activity and was partially rescued by a knockin mouse model of elevated Cdk1 activity. Additionally, we detected expression of Emi2 in spermatids and sperm, suggesting potential post-meiotic functions for Emi2.
- Published
- 2017
194. Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo
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Kathrin Klingner, Marie Follo, Julia Schüler, Monika Engelhardt, Dominik Schnerch, Dagmar Wider, Justus Duyster, Julia Felthaus, Ralph Wäsch, and Christine Greil
- Subjects
0301 basic medicine ,Cyclin B ,Cell Cycle Proteins ,Bortezomib ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,mitotic slippage ,AML ,Mice, Inbred NOD ,hemic and lymphatic diseases ,Antineoplastic Combined Chemotherapy Protocols ,APC/C ,proteasome inhibition ,Hematology ,biology ,Pteridines ,Cytarabine ,Myeloid leukemia ,Volasertib ,Leukemia, Myeloid, Acute ,Treatment Outcome ,Oncology ,030220 oncology & carcinogenesis ,Proteasome Inhibitors ,medicine.drug ,Research Paper ,medicine.medical_specialty ,Frail Elderly ,Mitosis ,Protein Serine-Threonine Kinases ,03 medical and health sciences ,Internal medicine ,Cell Line, Tumor ,Proto-Oncogene Proteins ,medicine ,Animals ,Humans ,neoplasms ,Protein Kinase Inhibitors ,Aged ,business.industry ,Xenograft Model Antitumor Assays ,Transplantation ,030104 developmental biology ,chemistry ,antimitotic therapy ,Immunology ,biology.protein ,Cancer research ,business - Abstract
// Dominik Schnerch 1 , Julia Schuler 2 , Marie Follo 1 , Julia Felthaus 1 , Dagmar Wider 1 , Kathrin Klingner 2 , Christine Greil 1 , Justus Duyster 1 , Monika Engelhardt 1 , Ralph Wasch 1 1 Department of Hematology, Oncology and Stem Cell Transplantation, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany 2 Oncotest GmbH, Freiburg, Germany Correspondence to: Ralph Wasch, email: ralph.waesch@uniklinik-freiburg.de Keywords: AML, antimitotic therapy, APC/C, mitotic slippage, proteasome inhibition Received: December 22, 2016 Accepted: February 07, 2017 Published: February 18, 2017 ABSTRACT Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.
- Published
- 2017
195. Coactivator functions in a stoichiometric complex with anaphase-promoting complex/cyclosome to mediate substrate recognition.
- Author
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Passmore, Lori A. and Barford, David
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LIGASES ,ENZYMES ,UBIQUITIN ,PROTEINS ,CYCLINS ,GROWTH factors ,CELL cycle ,BIOLOGICAL rhythms - Abstract
The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ligase required for ubiquitin-dependent proteolysis of cell-cycle-regulatory proteins, including mitotic cyclins and securin/Pds1. Regulation of APC/C activity and substrate recognition, mediated by the coactivators Cdc20 and Cdh1, is fundamental to cell-cycle control. However, the precise mechanism by which coactivators stimulate APC/C ubiquitylation activity and the nature of the substrate-binding sites on the activated APC/C are not understood. Here, we show that the optimal interaction of substrate with APC/C is dependent specifically on the simultaneous association of coactivator. This is consistent with a model whereby both core APC/C subunits and coactivators contribute recognition sites for substrates, accounting for the bipartite nature (D and KEN boxes) of most APC/C degradation signals. A direct and stoichiometric function for the coactivators could explain how specific substrates are recognized by APC/C in a cell-cycle-specific manner, and how coactivator stimulates APC/C ubiquitylation activity. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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196. Control of dTTP pool size by anaphase promoting complex/cyclosome is essential for the maintenance of genetic stability.
- Author
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Po-Yuan Ke, Yuan-Yeh Kuo, Chuan-Mei Hu, and Zee-Fen Chang
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PROTEOLYSIS , *CELL cycle , *THYMIDINE , *MITOSIS , *GROWTH factors - Abstract
Anaphase promoting complex/cyclosome (APC/C)-mediated proteolysis is essential for chromosome segregation, mitotic exit, and G1 entry. Here, we show the importance of APC/C in the control of dTTP pool size in mammalian cells. Two enzymes, thymidine kinase 1 (TK1) and thymidylate kinase (TMPK), involved in dTTP formation are the targets of the APC/C pathway. We demonstrate that TMPK is recognized and degraded by APC/C-Cdc20/Cdhl-mediated pathways from mitosis to the early G1 phase, whereas TK1 is targeted for degradation by APC/C-Cdhl after mitotic exit. Overexpression of wild-type TK1 and TMPK induces a four- to fivefold increase in the cellular dTTP pool without promoting spontaneous mutations in the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene. In contrast, coexpression of nondegradable TK1 and TMPK expands the dTTP pool size 10-fold accompanied by a drastic dNTP pool imbalance. Most interestingly, disruption of APC/C proteolysis of TK1 and TMPK leads to growth retardation and a striking increase in gene mutation rate. We conclude that down-regulation of dTTP pool size by the APC/C pathway during mitosis and the G1 phase is an essential means to maintain a balanced dNTP pool and to avoid genetic instability. [ABSTRACT FROM AUTHOR]
- Published
- 2005
197. A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin
- Author
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Willems, Andrew R., Schwab, Michael, and Tyers, Mike
- Subjects
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LEAVENING agents , *ENZYMES , *CELL growth , *CELL proliferation , *TRANSCRIPTION factors - Abstract
Abstract: The SCF (Skp1–Cullin–F-box) E3 ubiquitin ligase family was discovered through genetic requirements for cell cycle progression in budding yeast. In these multisubunit enzymes, an invariant core complex, composed of the Skp1 linker protein, the Cdc53/Cul1 scaffold protein and the Rbx1/Roc1/Hrt1 RING domain protein, engages one of a suite of substrate adaptors called F-box proteins that in turn recruit substrates for ubiquitination by an associated E2 enzyme. The cullin–RING domain–adaptor architecture has diversified through evolution, such that in total many hundreds of distinct SCF and SCF-like complexes enable degradation of myriad substrates. Substrate recognition by adaptors often depends on posttranslational modification of the substrate, which thus places substrate stability under dynamic regulation by intracellular signaling events. SCF complexes control cell proliferation through degradation of critical regulators such as cyclins, CDK inhibitors and transcription factors. A plethora of other processes in development and disease are controlled by other SCF-like complexes, including those based on Cul2–SOCS-box adaptor protein and Cul3–BTB domain adaptor protein combinations. Recent structural insights into SCF-like complexes have begun to illuminate aspects of substrate recognition and catalytic reaction mechanisms. [Copyright &y& Elsevier]
- Published
- 2004
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198. Caenorhabditis elegans UBC-2 functions with the anaphase-promoting but also has other activities.
- Author
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Frazier, Tyralynn, Shakes, Diane, Hota, Uma, and Boyd, Lynne
- Subjects
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PROTEINS , *AMINO acids , *CHEMICAL reactions , *RADIOACTIVITY , *CELL membranes , *RESONANCE , *CELL communication - Abstract
β-Secretase (BACE) carries out the first of two proteolysis steps to generate the amyloid-β peptides that accumulate in the senile plaques in Alzheimer's disease (AD). Because most BACE activity occurs in endosomes, signals regulating its trafficking to these compartments are important to an understanding of AD pathogenesis. A DISLL sequence near the BACE C-terminus mediates binding of BACE to the VHS domains of Golgi-localized gg-ear-containing ARF-binding (GGA) proteins, which are involved in the sorting of proteins to endosomes. Phosphorylation of the motif's serine residue regulates BACE recycling back to the cell surface from early endosomes and enhances the interaction of BACE with GGA proteins in isolated protein assays. We found that BACE phosphorylation influences BACE-GGA interactions in cells using a new fluorescence-resonance-energy-transfer-based assay of protein proximity, fluorescence lifetime imaging. Although serine-phosphorylated BACE was distributed throughout the cell, interaction of GGA1 with the wild-type protein occurred in juxtanuclear compartments. Pseudo-phosphorylated and non-phosphorylated BACE mutants remained localized with GGA1 in the Golgi body, but the latter mutation diminished the two proteins' FRET signal. Because BACE phosphorylated at serine residues can be identified in human brain, these data suggest that serine phosphorylation of BACE is a physiologically relevant posttranslational modification that regulates trafficking in the juxtanuclear compartment by interaction with GEA1. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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199. Mammalian Cdh1/Fzr mediates its own degradation.
- Author
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Listovsky, Tamar, Oren, Yifat S., Yudkovsky, Vana, Mahbubani, Hiro M., Weiss, Aryeh M., Lebendiker, Mario, and Brandeis, Michael
- Subjects
- *
CELL cycle , *BIOLOGICAL rhythms , *PROTEINS , *UBIQUITIN , *CELL proliferation , *XENOPUS , *PIPIDAE - Abstract
The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
200. The spindle checkpoint, aneuploidy, and cancer.
- Author
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Bharadwaj, Rajnish and Yu, Hongtao
- Subjects
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CHROMOSOMES , *CANCER cells , *CHROMOSOME abnormalities , *ANEUPLOIDY , *MITOSIS - Abstract
Cancer cells contain abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Defects in a cell cycle surveillance mechanism called the spindle checkpoint contribute to chromosome instability and aneuploidy. In response to straying chromosomes in mitosis, the spindle checkpoint inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C), thus preventing precocious chromosome segregation and ensuring the accurate partition of the genetic material. We review recent progress toward the understanding of the molecular mechanism of the spindle checkpoint and its role in guarding genome integrity at the chromosome level.Oncogene (2004) 23, 2016-2027. doi:10.1038/sj.onc.1207374 [ABSTRACT FROM AUTHOR]
- Published
- 2004
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