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Toru Miyazaki - One of the best experts on this subject based on the ideXlab platform.
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death effector domain containing protein dedd is an inhibitor of mitotic cdk1 Cyclin B1
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Satoko Arai, Shino Nemoto, Toru Miyazaki, Edward K. Wakeland, Katsuhisa Miyake, Renate Voit, Ingrid GrummtAbstract:Accumulating evidence has shown that many molecules, including some Cyclin-dependent kinases (Cdks) and Cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/Cyclin B1 complex. DEDD associates with mitotic Cdk1/Cyclin B1 complexes via direct binding to Cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/Cyclin B1 in DEDD-null (DEDD−/−) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD−/− cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD−/− mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
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Death-effector domain-containing protein DEDD is an inhibitor of mitotic Cdk1/Cyclin B1
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Satoko Arai, Shino Nemoto, Edward K. Wakeland, Katsuhisa Miyake, Renate Voit, Ingrid Grummt, Toru MiyazakiAbstract:Accumulating evidence has shown that many molecules, including some Cyclin-dependent kinases (Cdks) and Cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/Cyclin B1 complex. DEDD associates with mitotic Cdk1/Cyclin B1 complexes via direct binding to Cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/Cyclin B1 in DEDD-null (DEDD−/−) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD−/− cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD−/− mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
Jonathon Pines - One of the best experts on this subject based on the ideXlab platform.
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Cyclin B1 is essential for mitosis in mouse embryos and its nuclear export sets the time for mitosis
Journal of Cell Biology, 2018Co-Authors: Bernhard Strauss, Andrew Harrison, Paula A Coelho, Keiko Yata, Magdalena Zernickagoetz, Jonathon PinesAbstract:There is remarkable redundancy between the Cyclin–Cdk complexes that comprise the cell cycle machinery. None of the mammalian A-, D-, or E-type Cyclins are required in development until implantation, and only Cdk1 is essential for early cell divisions. Cyclin B1 is essential for development, but whether it is required for cell division is contentious. Here, we used a novel imaging approach to analyze Cyclin B1–null embryos from fertilization onward. We show that Cyclin B1−/− embryos arrest in G2 phase after just two divisions. This is the earliest arrest of any Cyclin known and places Cyclin B1 with cdk1 as the essential regulators of the cell cycle. We reintroduced mutant proteins into this genetically null background to determine why Cyclin B1 is constantly exported from the nucleus. We found that Cyclin B1 must be exported from the nucleus for the cell to prevent premature entry to mitosis, and retaining Cyclin B1–Cdk1 at the plasma membrane precludes entry to mitosis.
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activation of Cyclin B1 cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis
Journal of Cell Biology, 2010Co-Authors: Olivier Gavet, Jonathon PinesAbstract:The Cyclin B–Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, Cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of Cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export. In this study, we use a biosensor specific for Cyclin B1–Cdk1 activity to show that activating Cyclin B1–Cdk1 immediately triggers its rapid accumulation in the nucleus through a 40-fold increase in nuclear import that remains dependent on Cdk1 activity until NEBD. Nevertheless, a substantial proportion of Cyclin B1–Cdk1 remains in the cytoplasm. The increase in nuclear import is driven by changes in the nuclear import machinery that require neither Plk1 nor inhibition of nuclear export. Thus, the intrinsic link between Cyclin B1–Cdk1 activation and its rapid nuclear import inherently coordinates the reorganization of the nucleus and the cytoplasm at mitotic entry.
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active Cyclin B1 cdk1 first appears on centrosomes in prophase
Nature Cell Biology, 2003Co-Authors: Mark Jackman, Catherine Lindon, Erich A Nigg, Jonathon PinesAbstract:Cyclin B1–Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm, as just before nuclear envelope breakdown, Polo-like kinase1 (Plk1) is proposed to phosphorylate Cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. We raised phospho-specific antibodies against Cyclin B1 that primarily recognise the active form of the complex. We show that Cyclin B1 is initially phosphorylated on centrosomes in prophase and that Plk1 phosphorylates Cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause Cyclin B1 to move into the nucleus. We conclude that Cyclin B1–Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis.
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translocation of Cyclin B1 to the nucleus at prophase requires a phosphorylation dependent nuclear import signal
Current Biology, 1999Co-Authors: Anja Hagting, Mark Jackman, Karen Simpson, Jonathon PinesAbstract:Abstract Background: At M phase, Cyclin B1 is phosphorylated in the cytoplasmic retention sequence (CRS), which is required for nuclear export. During interphase, Cyclin B1 shuttles between the nucleus and the cytoplasm because constitutive nuclear import is counteracted by rapid nuclear export. In M phase, Cyclin B moves rapidly into the nucleus coincident with its phosphorylation, an overall movement that might be caused simply by a decrease in its nuclear export. However, the questions of whether CRS phosphorylation is required for Cyclin B1 translocation in mitosis and whether a reduction in nuclear export is sufficient to explain its rapid relocalisation have not been addressed. Results: We have used two forms of green fluorescent protein to analyse simultaneously the translocation of wild-type Cyclin B1 and a phosphorylation mutant of Cyclin B1 in mitosis, and correlated this with an in vitro nuclear import assay. We show that Cyclin B1 rapidly translocates into the nucleus approximately 10 minutes before breakdown of the nuclear envelope, and that this movement requires the CRS phosphorylation sites. A Cyclin B1 mutant that cannot be phosphorylated enters the nucleus after the wild-type protein. Phosphorylation of the CRS creates a nuclear import signal that enhances Cyclin B1 import in vitro and in vivo , in a manner distinct from the previously described import of Cyclin B1 mediated by importin β. Conclusions: We show that phosphorylation of human Cyclin B1 is required for its rapid translocation to the nucleus towards the end of prophase. Phosphorylation enhances Cyclin B1 nuclear import by creating a nuclear import signal. The phosphorylation of the CRS is therefore a critical step in the control of mitosis.
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temporal and spatial control of Cyclin B1 destruction in metaphase
Nature Cell Biology, 1999Co-Authors: Paul Clute, Jonathon PinesAbstract:The proteolysis of key regulatory proteins is thought to control progress through mitosis. Here we analyse Cyclin B1 degradation in real time and find that it begins as soon as the last chromosome aligns on the metaphase plate, just after the spindle-assembly checkpoint is inactivated. At this point, Cyclin B1 staining disappears from the spindle poles and from the chromosomes. Cyclin B1 destruction can subsequently be inactivated throughout metaphase if the spindle checkpoint is reimposed, and this correlates with the reappearance of Cyclin B1 on the spindle poles and the chromosomes. These results provide a temporal and spatial link between the spindle-assembly checkpoint and ubiquitin-mediated proteolysis.
Joel D Richter - One of the best experts on this subject based on the ideXlab platform.
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cpeb maskin and Cyclin B1 mrna at the mitotic apparatus implications for local translational control of cell division
Cell, 2000Co-Authors: Irina Groisman, Yishuian Huang, Raul Mendez, Quiping Cao, William E Theurkauf, Joel D RichterAbstract:In Xenopus development, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. CPEB and maskin, two factors that control polyadenylation-induced translation are present on the mitotic apparatus of animal pole blastomeres in embryos. Cyclin B1 protein and mRNA, whose translation is regulated by polyadenylation, are colocalized with CPEB and maskin. CPEB interacts with microtubules and is involved in the localization of Cyclin B1 mRNA to the mitotic apparatus. Agents that disrupt polyadenylation-induced translation inhibit cell division and promote spindle and centrosome defects in injected embryos. Two of these agents inhibit the synthesis of Cyclin B1 protein and one, which has little effect on this process, disrupts the localization of Cyclin B1 mRNA and protein. These data suggest that CPEB-regulated mRNA translation is important for the integrity of the mitotic apparatus and for cell division.
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the control of Cyclin B1 mrna translation during mouse oocyte maturation
Developmental Biology, 2000Co-Authors: Joyce Tay, Rebecca Hodgman, Joel D RichterAbstract:In maturing mouse oocytes, protein synthesis is required for meiotic maturation subsequent to germinal vesicle breakdown (GVBD). While the number of different proteins that must be synthesized for this progression to occur is unknown, at least one of them appears to be Cyclin B1, the regulatory subunit of M-phase-promoting factor. Here, we investigate the mechanism of Cyclin B1 mRNA translational control during mouse oocyte maturation. We show that the U-rich cytoplasmic polyadenylation element (CPE), a cis element in the 3′ UTR of Cyclin B1 mRNA, mediates translational repression in GV-stage oocytes. The CPE is also necessary for cytoplasmic polyadenylation, which stimulates translation during oocyte maturation. The injection of oocytes with a Cyclin B1 antisense RNA, which probably precludes the binding of a factor to the CPE, delays cytoplasmic polyadenylation as well as the transition from GVBD to metaphase II. CPEB, which interacts with the Cyclin B1 CPE and is present throughout meiotic maturation, becomes phosphorylated at metaphase I. These data indicate that CPEB is involved in both the repression and the stimulation of Cyclin B1 mRNA and suggest that the phosphorylation of this protein could be involved in regulating its activity.
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cytoplasmic polyadenylation elements mediate masking and unmasking of Cyclin B1 mrna
The EMBO Journal, 1999Co-Authors: Cornelia H De Moor, Joel D RichterAbstract:During oocyte maturation, Cyclin B1 mRNA is translationally activated by cytoplasmic polyadenylation. This process is dependent on cytoplasmic polyadenylation elements (CPEs) in the 3′ untranslated region (UTR) of the mRNA. To determine whether a titratable factor might be involved in the initial translational repression (masking) of this mRNA, high levels of Cyclin B1 3′ UTR were injected into oocytes. While this treatment had no effect on the poly(A) tail length of endogenous Cyclin B1 mRNA, it induced Cyclin B1 synthesis. A mutational analysis revealed that the most efficient unmasking element in the Cyclin 3′ UTR was the CPE. However, other U‐rich sequences that resemble the CPE in structure, but which do not bind the CPE‐binding polyadenylation factor CPEB, failed to induce unmasking. When fused to the chloramphenical acetyl transferase (CAT) coding region, the Cyclin B1 3′ UTR inhibited CAT translation in injected oocytes. In addition, a synthetic 3′ UTR containing multiple copies of the CPE also inhibited translation, and did so in a dose‐dependent manner. Furthermore, efficient CPE‐mediated masking required cap‐dependent translation. During the normal course of progesterone‐induced maturation, cytoplasmic polyadenylation was necessary for mRNA unmasking. A model to explain how Cyclin B1 mRNA masking and unmasking could be regulated by the CPE is presented.
David O Morgan - One of the best experts on this subject based on the ideXlab platform.
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structural basis of human separase regulation by securin and cdk1 Cyclin B1
Nature, 2021Co-Authors: Pierre Raia, David O Morgan, Chloe M Ghent, Tobias Raisch, Yashar Sadian, Simone Cavadini, Pramod M Sabale, David Barford, Stefan Raunser, Andreas BolandAbstract:In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase—a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212–4). Separase is activated by degradation of its inhibitors, securin5 and Cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1–Cyclin B1–CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1–Cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1–Cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in Cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1–Cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation. Structures of separase in complex with either securin or Cyclin B–CDK1 shed light on the regulation of chromosome separation during the cell cycle.
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control of mitosis by changes in the subcellular location of Cyclin B1 cdk1 and cdc25c
Current Opinion in Cell Biology, 2000Co-Authors: Catherine G Takizawa, David O MorganAbstract:Nuclear events of mitosis are initiated when the protein kinase Cyclin-B1-Cdk1 is translocated into the nucleus during prophase. Recent work has unveiled many of the mechanisms that govern the localization of Cyclin-B1-Cdk1 and its regulator Cdc25C. Phosphorylation-dependent changes in the rate of nuclear import and export of these proteins help to control the onset of mitosis both in normal cells and in cells delayed before mitosis by DNA damage.
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ran independent nuclear import of Cyclin B1 cdc2 by importin beta
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Catherine G Takizawa, Karsten Weis, David O MorganAbstract:Mitosis is triggered in vertebrate cells by the Cyclin B1–Cdc2 complex. The activation of this complex at the end of G2 phase is accompanied by its translocation from the cytoplasm to the nucleus. We used digitonin-permeabilized human cells to analyze the mechanism by which Cyclin B1–Cdc2 is imported into the nucleus. Cyclin B1–Cdc2 import was not blocked by inhibitors of the importin α-dependent import pathway or by dominant negative versions of the GTPase Ran or importin β. However, the rate of Cyclin B1 import was decreased by immunodepletion of importin β from cytosol. Purified importin β promoted Cyclin B1 import in the absence of cytosol or Ran and in the presence of the dominant negative Ran mutant. We conclude that Cyclin B1 import is mediated by an unusual importin β-dependent mechanism that does not require Ran.
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nuclear localization of Cyclin B1 controls mitotic entry after dna damage
Journal of Cell Biology, 1998Co-Authors: Pei Jin, Stephen Hardy, David O MorganAbstract:Mitosis in human cells is initiated by the protein kinase Cdc2-Cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-Cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear Cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear Cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of Cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear Cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of Cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.
Satoko Arai - One of the best experts on this subject based on the ideXlab platform.
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death effector domain containing protein dedd is an inhibitor of mitotic cdk1 Cyclin B1
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Satoko Arai, Shino Nemoto, Toru Miyazaki, Edward K. Wakeland, Katsuhisa Miyake, Renate Voit, Ingrid GrummtAbstract:Accumulating evidence has shown that many molecules, including some Cyclin-dependent kinases (Cdks) and Cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/Cyclin B1 complex. DEDD associates with mitotic Cdk1/Cyclin B1 complexes via direct binding to Cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/Cyclin B1 in DEDD-null (DEDD−/−) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD−/− cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD−/− mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
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Death-effector domain-containing protein DEDD is an inhibitor of mitotic Cdk1/Cyclin B1
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Satoko Arai, Shino Nemoto, Edward K. Wakeland, Katsuhisa Miyake, Renate Voit, Ingrid Grummt, Toru MiyazakiAbstract:Accumulating evidence has shown that many molecules, including some Cyclin-dependent kinases (Cdks) and Cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/Cyclin B1 complex. DEDD associates with mitotic Cdk1/Cyclin B1 complexes via direct binding to Cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/Cyclin B1 in DEDD-null (DEDD−/−) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD−/− cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD−/− mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
