scholarly journals The Anaphase-Promoting Complex/Cyclosome Activator Cdh1 Modulates Rho GTPase by Targeting p190 RhoGAP for Degradation

2010 ◽  
Vol 30 (16) ◽  
pp. 3994-4005 ◽  
Author(s):  
Hideaki Naoe ◽  
Kimi Araki ◽  
Osamu Nagano ◽  
Yusuke Kobayashi ◽  
Jo Ishizawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Rho Gtpase ◽  
Thrombus Formation ◽  
Fiber Formation ◽  
Anaphase Promoting Complex ◽  
Migration Activity ◽  
Eyelid Closure

ABSTRACT Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G1 maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1 GT/GT) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1 GT/GT MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1 GT/GT embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1 GT/GT embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.

scholarly journals Swm1/Apc13 Is an Evolutionarily Conserved Subunit of the Anaphase-Promoting Complex Stabilizing the Association of Cdc16 and Cdc27

2004 ◽  
Vol 24 (8) ◽  
pp. 3562-3576 ◽  
Author(s):  
Martin Schwickart ◽  
Jan Havlis ◽  
Bianca Habermann ◽  
Aliona Bogdanova ◽  
Alain Camasses ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Small Subunit ◽  
Cell Cycle Regulators ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Ligase Activity ◽  
Evolutionarily Conserved ◽  
Stable Association

ABSTRACT The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.

scholarly journals A Novel Pharmacological Method to Study the Chinese Medicinal Formula Hua-Zheng-Hui-Sheng-Dan

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Rui Cao ◽  
Hong Zhang ◽  
Jie Guo ◽  
Xiao-hui Liu ◽  
Chang Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Experimental Design ◽  
Molecular Biology ◽  
Antitumor Activity ◽  
Cell Viability ◽  
Hela Cells ◽  
Orthogonal Experimental Design ◽  
Original Formula

Objectives. Hua-Zheng-Hui-Sheng-Dan (HZHSD) was used as an experimental model to explore research methods of large formulae in traditional Chinese medicine (TCM) using current molecular biology approaches.Materials and Methods. The trypan blue exclusion assay was used to determine cell viability and cell numbers. Flow cytometry was used to assess cell cycle distribution and apoptosis. The concentration of cyclin D1 was analyzed by enzyme-linked immunosorbent assay. The median effect principle was used in drug combination studies. An orthogonal experimental design was used to estimate the effects of each herb at different concentrations. The HeLa xenograft mouse model was used to compare the antitumor activity of drugs in vivo.Results. Among the 35 herbs that comprise HZHSD, Radix Rehmanniae Preparata (RRP),Caesalpinia sappan(CS),Evodia rutaecarpa(ER), Folium Artemisiae Argyi (FAA),Leonurus japonicusHoutt (LJH), Tumeric (Tu), Radix Paeoniae Alba (RPA), and Trogopterus Dung (TD) effectively inhibited the proliferation of HeLa and SKOV3 cells. Only RRR had an effect on HeLa and SKOV3 cell viability. According to the median effect principle,Angelica sinensis(Oliv.) (AS),Tabanus(Ta), and Pollen Typhae (PT), which were proven to have a significant synergistic inhibitory effect on the proliferation of HeLa cells, were added to the original eight positive herbs. The combination of RPA and AS had a synergistic effect on inducing cell cycle S phase arrest and decreasing intracellular cyclin D1 in HeLa cells. By orthogonal experimental design, LJH and Tu were considered unnecessary herbs. The small formula (SHZHSD) consisted of RPA, AS, RRR, Ta., TD, PT, ER, CS, and FAA and was able to inhibit cell proliferation and induce cell apoptosis. The antitumor effects of HZHSD and SHZHSD were also compared in vivo.Conclusions. Through molecular biology approaches both in vitro and in vivo, research into single drugs, and analysis using the median effect principle and orthogonal experimental design, the small formula (SHZHSD) was determined from the original formula (HZHSD). SHZHSD exhibited superior antitumor activity compared with the original formula both in vitro and in vivo.

scholarly journals Pseudosubstrate Inhibition of the Anaphase-Promoting Complex by Acm1: Regulation by Proteolysis and Cdc28 Phosphorylation

2008 ◽  
Vol 28 (15) ◽  
pp. 4653-4664 ◽  
Author(s):  
Denis Ostapenko ◽  
Janet L. Burton ◽  
Ruiwen Wang ◽  
Mark J. Solomon
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Anaphase Promoting Complex ◽  
Inhibitory Protein ◽  
Ubiquitin Ligase Activity ◽  
Dependent Protein ◽  
Apc Mutation

ABSTRACT The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.

scholarly journals Phosphorylation of the Anaphase Promoting Complex activator CDH1/FZR regulates the transition from Meiosis I to Meiosis II in mouse male germ cell

2020 ◽  
Author(s):  
Nobuhiro Tanno ◽  
Shinji Kuninaka ◽  
Sayoko Fujimura ◽  
Kaho Okamura ◽  
Kazumasa Takemoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Germ Cells ◽  
Critical Role ◽  
Biological Significance ◽  
Mitotic Cell ◽  
Anaphase Promoting Complex ◽  
Meiosis I

SummaryCDH1/FZR is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of CDH1 during mitotic cell cycle. Although the critical role of CDH1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of CDH1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of CDH1. Although ablation of CDH1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of CDH1 phosphorylation, male juvenile germ cells entered meiosis normally but skipped meiosis II producing diploid spermatid-like cells. In aged testis, male germ cells were overall abolished, showing Sertoli cell-only phenotype. The present study demonstrated that phosphorylation of CDH1 is required for temporal regulation of APC/C activity at the transition from meiosis I to meiosis II, and for spermatoginial stem cell maintenance, which raised an insight into the sexual dimorphism of CDH1-regulation in germ cells.

scholarly journals Polyanions provide selective control of APC/C interactions with the activator subunit

2019 ◽  
Author(s):  
Arda Mizrak ◽  
David O. Morgan
Keyword(s):  
Cell Cycle ◽  
Nucleic Acids ◽  
Substrate Affinity ◽  
Anaphase Promoting Complex ◽  
Inhibitory Effects ◽  
Dissociation Kinetics ◽  
High Affinity ◽  
Transient Interactions

ABSTRACTTransient interactions between the Anaphase-Promoting Complex/Cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitination activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with the ubiquitination of low-affinity substrates. However, engagement with high-affinity substrates blocks the inhibitory effects of polyanions on activator binding and APC/C activity. This mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitination of high-affinity substrates and suppressing ubiquitination of low-affinity substrates.

scholarly journals Anaphase-Promoting Complex/Cyclosome Controls the Stability of TPX2 during Mitotic Exit

2005 ◽  
Vol 25 (23) ◽  
pp. 10516-10527 ◽  
Author(s):  
Scott Stewart ◽  
Guowei Fang
Keyword(s):  
Cell Cycle ◽  
Spindle Assembly ◽  
Mitotic Exit ◽  
Anaphase Promoting Complex ◽  
Discrete Elements ◽  
Protein Levels ◽  
Recognition Motifs ◽  
The Stability

ABSTRACT TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/CCdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/CCdh1 in vitro and for its degradation in vivo. We conclude that APC/CCdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.

scholarly journals Cell Cycle–regulated Proteolysis of Mitotic Target Proteins

1999 ◽  
Vol 10 (11) ◽  
pp. 3927-3941 ◽  
Author(s):  
Holger Bastians ◽  
Leana M. Topper ◽  
Gary L. Gorbsky ◽  
Joan V. Ruderman
Keyword(s):  
Cell Cycle ◽  
Metaphase Plate ◽  
S Phase ◽  
Dominant Negative ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Mitotic Cyclin ◽  
Ubiquitin Conjugating Enzyme

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase–anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C–dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1–S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

scholarly journals Polyanions provide selective control of APC/C interactions with the activator subunit

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Arda Mizrak ◽  
David O. Morgan
Keyword(s):  
Cell Cycle ◽  
Nucleic Acids ◽  
Substrate Affinity ◽  
Anaphase Promoting Complex ◽  
Inhibitory Effects ◽  
Dissociation Kinetics ◽  
High Affinity ◽  
Transient Interactions

AbstractTransient interactions between the anaphase-promoting complex/cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitylation activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with substrate ubiquitylation. However, engagement with high-affinity substrate blocks the inhibitory effects of polyanions on activator binding and APC/C activity. We propose that this mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitylation of high-affinity substrates and suppressing ubiquitylation of low-affinity substrates.

scholarly journals Human Menstrual Blood-Derived Stem Cells Inhibit the Proliferation of HeLa Cells via TGF-β1-Mediated JNK/P21 Signaling Pathways

2019 ◽  
Vol 2019 ◽  
pp. 1-18
Author(s):  
Qian-Yu Liu ◽  
Feng Ruan ◽  
Jing-Yuan Li ◽  
Li Wei ◽  
Ping Hu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Stem Cells ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Hela Cells ◽  
Cycle Arrest ◽  
Cervical Cancer Cells

Human menstrual blood-derived stem cells (hMBSCs) are a novel type of mesenchymal stem cells (MSCs) that have a high proliferative rate, multilineage differentiation potential, low immunogenicity, and low oncogenicity, making them suitable candidates for regenerative medicine. The therapeutic efficacy of hMBSCs has been demonstrated in some diseases; however, their effects on cervical cancer remain unclear. In the present study, we investigated whether hMBSCs have anticancer properties on cervical cancer cells in vivo and in vitro, which has not yet been reported. In vitro, transwell coculturing experiments revealed that hMBSCs suppress the proliferation and invasion of HeLa cervical cancer cells by inducing G0/G1 cell cycle arrest. In vivo, we established a xenografted BALB/c nude mouse model by subcutaneously coinjecting HeLa cells with hMBSCs for 21 days. We found that hMBSCs significantly decrease the average volume and average weight of xenografted tumors. ELISA, TGF-β1 antibody, and recombinant human TGF-β1 (rhTGF-β1) were used to analyze whether TGF-β1 contributed to cell cycle arrest. We found that hMBSC-secreted TGF-β1 and rhTGF-β1 induced cell cycle arrest and increased the expression of phospho-JNK and phospho-P21 in HeLa cells, which was mostly reversed by TGF-β1 antibody. These results indicate that hMBSCs have antitumor properties on cervical cancer in vitro and in vivo, mediated by the TGF-β1/JNK/p21 signaling pathway. In conclusion, this study suggests that hMBSC-based therapy is promising for the treatment of cervical cancer.

scholarly journals A Conserved Cyclin-Binding Domain Determines Functional Interplay between Anaphase-Promoting Complex–Cdh1 and Cyclin A-Cdk2 during Cell Cycle Progression

2001 ◽  
Vol 21 (11) ◽  
pp. 3692-3703 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mechanistic Explanation ◽  
Cyclin A ◽  
Binding Motif ◽  
Cell Cycle Regulators ◽  
Anaphase Promoting Complex ◽  
Cycle Progression

ABSTRACT Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G1/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1–cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G1/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.

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