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1.
Iwata E Ikeda S Matsunaga S Kurata M Yoshioka Y Criqui MC Genschik P Ito M 《The Plant cell》2011,23(12):4382-4393
Increased cellular ploidy is widespread during developmental processes of multicellular organisms, especially in plants. Elevated ploidy levels are typically achieved either by endoreplication or endomitosis, which are often regarded as modified cell cycles that lack an M phase either entirely or partially. We identified GIGAS CELL1 (GIG1)/OMISSION OF SECOND DIVISION1 (OSD1) and established that mutation of this gene triggered ectopic endomitosis. On the other hand, it has been reported that a paralog of GIG1/OSD1, UV-INSENSITIVE4 (UVI4), negatively regulates endoreplication onset in Arabidopsis thaliana. We showed that GIG1/OSD1 and UVI4 encode novel plant-specific inhibitors of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. These proteins physically interact with APC/C activators, CDC20/FZY and CDH1/FZR, in yeast two-hybrid assays. Overexpression of CDC20.1 and CCS52B/FZR3 differentially promoted ectopic endomitosis in gig1/osd1 and premature occurrence of endoreplication in uvi4. Our data suggest that GIG1/OSD1 and UVI4 may prevent an unscheduled increase in cellular ploidy by preferentially inhibiting APC/C(CDC20) and APC/C(FZR), respectively. Generation of cells with a mixed identity in gig1/osd1 further suggested that the APC/C may have an unexpected role for cell fate determination in addition to its role for proper mitotic progression. 相似文献
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Regulation of the Anaphase-promoting Complex/Cyclosome by
bimAAPC3 and Proteolysis of NIMA 下载免费PDF全文
Xiang S. Ye Russell R. Fincher Alice Tang Aysha
H. Osmani Stephen A. Osmani 《Molecular biology of the cell》1998,9(11):3019-3030
Surprisingly, although highly temperature-sensitive, the bimA1APC3 anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1APC3 is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34cdc2 kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1APC3-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1APC3 double mutant arrests in a mitotic state with very high p34cdc2 H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1APC3 mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1APC3. The bimA1APC3 mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner. 相似文献
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Objective: Chromosome segregation during mitosis requires a physically large proteinaceous structure called the kinetochore to generate attachments between chromosomal DNA and spindle microtubules. It is essential for kinetochore components to be carefully regulated to guarantee successful cell division. Depletion, mutation or dysregulation of kinetochore proteins results in mitotic arrest and/or cell death. HEC1 (high expression in cancer) has been reported to be a kinetochore protein, depletion of which, by RNA interference, results in catastrophic mitotic exit. Materials and methods and results: To investigate how HEC1 protein is controlled post‐translation, we analysed the role of anaphase‐promoting complex/cyclosome (APC/C)‐Cdh1 in degradation of HEC1 protein. In this study, we show that HEC1 is an unstable protein and can be targeted by endogenous ubiquitin‐proteasome system in HEK293T cells. Results of RNA interference and in vivo ubiquitination assay indicated that HEC1 could be ubiquitinated and degraded by APC/C‐hCdh1 E3 ligase. The evolutionally conserved D‐box at the C‐terminus functioned as the degron of HEC1, destruction of which resulted in resistance to degradation mediated by APC/C‐Cdh1. Overexpression of non‐degradable HEC1 (D‐box destroyed) induced accumulation of cyclin B protein in vivo and triggered mitotic arrest. Conclusion: APC/C‐Cdh1 controls stability of HEC1, ensuring normal cell cycle progression. 相似文献
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The rap (retina aberrant in pattern) gene encodes the Fizzy-related protein (Fzr), which as an activator of the ubiquitin ligase complex; APC/C (anaphase promoting complex/cyclosome) facilitates the cell cycle stage-specific degradation of cyclins. Loss-of-function mutations in rap cause unscheduled accumulation of cyclin B in the developing eye imaginal disc, resulting in additional mitotic cycles and defective patterning of the developing Drosophila eye. Targeted mis-expression of rap/fzr in the eye primordial cells causes precocious cell cycle exit, and smaller primordial eye fields, which either eliminate or drastically reduce the size of the adult eye. Although mitosis is inhibited in the mis-expression animals, cells with abnormally large nuclei form tumor-like structures from continued endoreplication, cell growth and retinal differentiation. Interestingly, overexpression of Rap/Fzr in the eye primordia also increases the size of the antennal primordium resulting in the induction of ectopic antennae. These results suggest that Rap/Fzr plays an essential role in the timely exit of precursor cells from mitotic cycles and indicate that mechanisms that regulate cell cycle exit are critical during pattern formation and morphogenesis. 相似文献
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Eloy NB de Freitas Lima M Van Damme D Vanhaeren H Gonzalez N De Milde L Hemerly AS Beemster GT Inzé D Ferreira PC 《The Plant journal : for cell and molecular biology》2011,68(2):351-363
The largest E3 ubiquitin-ligase complex, known as anaphase-promoting complex/cyclosome (APC/C), regulates the proteolysis of cell cycle regulators such as CYCLIN B and SECURIN that are essential for sister-chromatid separation and exit from mitosis. Despite its importance, the role of APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, the Arabidopsis thaliana APC/C subunit APC10 was characterized and shown to functionally complement an apc10 yeast mutant. The APC10 protein was located in specific nuclear bodies, most probably resulting from its association with the proteasome complex. An apc10 Arabidopsis knockout mutant strongly impaired female gametogenesis. Surprisingly, constitutive overexpression of APC10 enhanced leaf size. Through kinematic analysis, the increased leaf size was found to be due to enhanced rates of cell division during the early stages of leaf development and, at the molecular level, by increased APC/C activity as measured by an amplification of the proteolysis rate of the mitotic cyclin, CYCB1;1. 相似文献
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The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. 相似文献
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Mariana Romeiro Motta XinAi Zhao Martine Pastuglia Katia Belcram Farshad Roodbarkelari Maki Komaki Hirofumi Harashima Shinichiro Komaki Manoj Kumar Petra Bulankova Maren Heese Karel Riha David Bouchez Arp Schnittger 《EMBO reports》2022,23(1)
Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development‐specific requirements of B1‐type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1‐type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1‐type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA‐TUBULIN COMPLEX PROTEIN 3‐INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1‐type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s. 相似文献
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Zemfira Karamysheva Laura A. Diaz-Martinez Sara E. Crow Bing Li Hongtao Yu 《The Journal of biological chemistry》2009,284(3):1772-1780
Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early
mitosis and, thus, prevents premature sister-chromatid separation. The protein
level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is
down-regulated in G1/S. Here we show that Sgo1 is degraded during
the exit from mitosis, and its degradation depends on the anaphase-promoting
complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels
of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C
bound to Cdh1 (APC/CCdh1) in vitro. We have further
identified two functional degradation motifs in Sgo1; that is, a KEN
(Lys-Glu-Asn) box and a destruction box (D box). Although removal of either
motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box
deleted is stable in cells. Surprisingly, mitosis progresses normally in the
presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not
required for sister-chromatid separation or mitotic exit. Finally, we show
that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1
steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in
mitosis and occurs in two steps in vertebrate cells
(1-3).
In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and
removed from chromosome arms
(1,
4). Then, cleavage of
centromeric cohesin by separase takes place at the metaphase-to-anaphase
transition to allow sister-chromatid separation
(5). The shugoshin (Sgo) family
of proteins plays an important role in the protection of centromeric cohesion
(6,
7). Human cells depleted of
Sgo1 by RNAi undergo massive chromosome missegregation
(8-11).
In cells with compromised Sgo1 function, centromeric cohesin is improperly
phosphorylated and removed (4,
11), resulting in premature
sister-chromatid separation. It has been shown recently that Sgo1 collaborates
with PP2A to counteract the action of Plk1 and other mitotic kinases and to
protect centromeric cohesin from premature removal
(12-14).
In addition, Sgo1 has also been shown to promote stable
kinetochore-microtubule attachment and sense tension across sister
kinetochores (8,
15). Thus, Sgo1 is crucial for
mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting
complex/cyclosome
(APC/C),2 a large
multiprotein ubiquitin ligase that targets key mitotic regulators for
destruction by the proteasome
(16). APC/C selects substrates
for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize
specific sequences called APC/C degrons within target proteins
(17). Several APC/C degrons
have been characterized, including the destruction box (D box) and the
Lys-Glu-Asn box (KEN box) (18,
19). The D box, with the
consensus amino acid sequence of RXXLXXXN(X
indicates any amino acid), are found in many APC/C substrates, including
mitotic cyclins and are essential for their ubiquitin-mediated destruction.
The KEN box, which contains a consensus KEN motif, is also found in several
APC/C substrates and is preferentially but not exclusively recognized by
APC/CCdh1. When APC/C is active, it directs progression through and
exit from mitosis by catalyzing the ubiquitination and timely destruction of
mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor
securin (16). The APC/C
activity needs to be tightly controlled to prevent unscheduled substrate
degradation. An important mechanism for APC/C regulation is the spindle
checkpoint, which prevents the activation of APC/C and destruction of its
substrates in response to kinetochores that have not properly attached to the
mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein
levels oscillate during the cell cycle
(8,
9). In this article we study
the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during
mitotic exit, and this degradation depends on APC/CCdh1. We further
show that both KEN and D boxes are required for Sgo1 degradation in
vivo and ubiquitination in vitro. Removal of these motifs
stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1
protein in human cells does not change the kinetics of chromosome segregation
and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes
place but is not required for mitotic exit. Finally, we show that Bub1
regulates Sgo1 protein levels through a mechanism that does not involve
APC/C-mediated degradation. 相似文献
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Disruption of early mitotic inhibitor 1 (Emi1) interferes with normal cell cycle progression and results in early embryonic lethality in vertebrates. During S and G2 phases the ubiquitin ligase complex APC/C is inhibited by Emi1 protein, thereby enabling the accumulation of Cyclins A and B so they can regulate replication and promote the transition from G2 phase to mitosis, respectively. Depletion of Emi1 prevents mitotic entry and causes rereplication and an increase in cell size. In this study, we show that the developmental and cell cycle defects caused by inactivation of zebrafish emi1 are due to inappropriate activation of APC/C through its cofactor Cdh1. Inhibiting/slowing progression into S-phase by depleting Cdt1, an essential replication licensing factor, partially rescued emi1 deficiency-induced rereplication and the increased cell size. The cell size effect was enhanced by co-depletion of cell survival regulator p53. These data suggest that the increased size of emi1-deficient cells is either directly or indirectly caused by the rereplication defects. Moreover, enforced expression of Cyclin A partially ablated the rereplicating population in emi1-deficient zebrafish embryos, consistent with the role of Cyclin A in origin licensing. Forced expression of Cyclin B partially restored the G1 population, in agreement with the established role of Cyclin B in mitotic progression and exit. However, expression of Cyclin B also partially inhibited rereplication in emi1-deficient embryos, suggesting a role for Cyclin B in regulating replication in this cellular context. As Cyclin A and B are substrates for APC/C-Cdh1 - mediated degradation, and Cdt1 is under control of Cyclin A, these data indicate that emi1 deficiency-induced defects in vivo are due to the dysregulation of an APC/C-Cdh1 molecular axis. 相似文献
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Himanshu Singh Vandana Raghavan Manish Shukla Basuthkar J. Rao Kandala V. R. Chary 《Biomolecular NMR assignments》2014,8(1):71-74
Almost complete sequence specific 1H, 13C and 15N resonance assignments of S114A mutant of UVI31+ from Chlamydomonas reinhardtii are reported. The cDNA of S114A mutant of UVI31+ was cloned from a eukaryotic green algae (C. reinhardtii) and overexpressed in E.coli from where the protein was purified to homogeneity. The point mutation S114A in UVI31+ reduces its DNA endonuclease activity substantially as compared with its wild type. As a prelude to the structural characterization of S114A mutant of UVI31+, we report here complete sequence-specific 1H, 13C and 15N NMR assignments. 相似文献
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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. 相似文献
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Kobi J Simpson-Lavy Drora Zenvirth Michael Brandeis 《Cell cycle (Georgetown, Tex.)》2015,14(19):3138-3145
The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/CCdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/CCdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation. 相似文献
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Olga Nagy Margit Pál Andor Udvardy Christine AM Shirras Imre Boros Alan D Shirras Péter Deák 《Cell division》2012,7(1):1-15
Background
The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.Results
Here we describe mitotic slippage in yeast bub2?? mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.Conclusions
The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans. 相似文献20.
Laura A. Fisher Ling Wang Lan Wu Aimin Peng 《The Journal of biological chemistry》2014,289(34):23745-23752
Mitotic progression is regulated largely through dynamic and reversible protein phosphorylation that is modulated by opposing actions of protein kinases and phosphatases. In this study, we show that phosphatase 1 nuclear targeting subunit (Pnuts) functions as a master regulator of mitosis by modulating protein phosphatase 1 (PP1). Overexpression of Pnuts in Xenopus egg extracts inhibited both mitotic and meiotic exit. Immunodepletion of Pnuts from egg extracts revealed its essential functions in mitotic entry and maintenance. The level of Pnuts oscillates during the cell cycle and peaks in mitosis. Pnuts destruction during M-phase exit is mediated by the anaphase-promoting complex/cyclosome (APC/C)-targeted ubiquitination and proteolysis, and conserved destruction motifs of Pnuts. Disruption of Pnuts degradation delayed M-phase exit, suggesting it as an important mechanism to permit M-phase exit. 相似文献