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

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.

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Upregulation of SGOL2 Predicts Poor Prognosis and Facilitates Hepatocellular Carcinoma Progression via Dysregulating the Cell Cycle by Directly Activating MAD2

10.21203/rs.3.rs-560609/v1 ◽

2021 ◽

Author(s):

Qingqing Hu ◽

Xiaochu Hu ◽

Yalei Zhao ◽

Lingjian Zhang ◽

Ya Yang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

The Cancer Genome Atlas ◽

Loss Of Function ◽

Protein Levels ◽

Cancer Genome Atlas ◽

Centromeric Protein ◽

Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Shugoshin-like protein 2 (SGOL2) is a centromeric protein that ensures the correct and orderly process of mitosis by protecting and maintaining centripetal adhesions during meiosis and mitosis. However, the role of SGOL2 in cancer is not well understood. Methods: The mRNA and protein levels of SGOL2 and survival analysis were conducted in The Cancer Genome Atlas (TCGA) and further validated in 2 independent cohorts. Differential genes correlated with SGOL2 and mitotic arrest deficient 2 like 1 (MAD2) were obtained using LinkedOmics. Subsequently, loss-of-function and rescue assays were carried out in vitro and in vivo to assess the functions of SGOL2 in hepatic tumorigenisis. Findings: We found that SGOL2 was significantly overexpressed in HCC and predicted unfavorable overall survival in HCC patients. Next, we identified 47 differentially expressed genes positively correlated with both SGOL2 and MAD2 to be mainly involved in the cell cycle. In addition, SGOL2 downregulation suppressed the migration, invasion, proliferation, stemness and EMT of HCC cells and inhibited tumorigenesis in vivo. Furthermore, SGOL2 promoted tumor proliferation by activating MAD2-induced cell cycle dysregulation, which could be reversed by the MAD2 inhibitor M2I-1. We also proved that SGOL2 activated MAD2 by directly binding with MAD2. Conclusions: The results of this study showed that SGOL2 acts as an oncogene in HCC cells by directly activating MAD2 and then dysregulating the cell cycle, thereby providing a potential target for HCC patients in the future.

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Effect and mechanism of YB-1 knockdown on glioma cell growth, migration, and apoptosis

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmz161 ◽

2020 ◽

Vol 52 (2) ◽

pp. 168-179 ◽

Cited By ~ 1

Author(s):

Huilin Gong ◽

Shan Gao ◽

Chenghuan Yu ◽

Meihe Li ◽

Ping Liu ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Growth ◽

Glioma Cell ◽

Cell Transformation ◽

Malignant Cell ◽

Protein Levels ◽

Glioma Progression

Abstract Y-box binding protein 1 (YB-1) is manifested as its involvement in cell proliferation and differentiation and malignant cell transformation. Overexpression of YB-1 is associated with glioma progression and patient survival. The aim of this study is to investigate the influence of YB-1 knockdown on glioma cell progression and reveal the mechanisms of YB-1 knockdown on glioma cell growth, migration, and apoptosis. It was found that the knockdown of YB-1 decreased the mRNA and protein levels of YB-1 in U251 glioma cells. The knockdown of YB-1 significantly inhibited cell proliferation, colony formation, and migration in vitro and tumor growth in vivo. Proteome and phosphoproteome data revealed that YB-1 is involved in glioma progression through regulating the expression and phosphorylation of major proteins involved in cell cycle, adhesion, and apoptosis. The main regulated proteins included CCNB1, CCNDBP1, CDK2, CDK3, ADGRG1, CDH-2, MMP14, AIFM1, HO-1, and BAX. Furthermore, it was also found that YB-1 knockdown is associated with the hypo-phosphorylation of ErbB, mTOR, HIF-1, cGMP-PKG, and insulin signaling pathways, and proteoglycans in cancer. Our findings indicated that YB-1 plays a key role in glioma progression in multiple ways, including regulating the expression and phosphorylation of major proteins associated with cell cycle, adhesion, and apoptosis.

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Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.24.2.514-526.2004 ◽

2004 ◽

Vol 24 (2) ◽

pp. 514-526 ◽

Cited By ~ 75

Author(s):

Po-Yuan Ke ◽

Zee-Fen Chang

Keyword(s):

Cell Cycle ◽

Thymidine Kinase ◽

Mammalian Cells ◽

Mitotic Exit ◽

Limiting Factor ◽

Anaphase Promoting Complex ◽

Thymidine Kinase 1 ◽

Ubiquitin Proteasome ◽

The Right

ABSTRACT The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G1 phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphase-promoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G1 phase of the cell cycle in mammalian cells.

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BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy

BioMed Research International ◽

10.1155/2021/5556306 ◽

2021 ◽

Vol 2021 ◽

pp. 1-19

Author(s):

Weiya Cao ◽

Xueke Liu ◽

Yinci Zhang ◽

Amin Li ◽

Yinghai Xie ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Cell Lines ◽

Acquired Resistance ◽

Inhibitory Effect ◽

Mtor Pathway ◽

Protein Levels ◽

Huh7 Cells

Acquired resistance of hepatocellular carcinoma (HCC) to sorafenib (SFB) is the main reason for the failure of SFB treatment of the cancer. Abnormal activation of the PI3K/AKT/mTOR pathway is important in the acquired resistance of SFB. Therefore, we investigated whether BEZ235 (BEZ) could reverse acquired sorafenib resistance by targeting the PI3K/mTOR pathway. A sorafenib-resistant HCC cell line Huh7R was established. MTT assay, clone formation assay, flow cytometry, and immunofluorescence were used to analyze the effects of BEZ235 alone or combined with sorafenib on cell proliferation, cell cycle, apoptosis, and autophagy of Huh7 and Huh7R cells. The antitumor effect was evaluated in animal models of Huh7R xenografts in vivo. Western blot was used to detect protein levels of the PI3K/AKT/mTOR pathway and related effector molecules. In vitro results showed that the Huh7R had a stronger proliferation ability and antiapoptosis effect than did Huh7, and sorafenib had no inhibitory effect on Huh7R. SFB + BEZ inhibited the activation of the PI3K/AKT/mTOR pathway caused by sorafenib. Moreover, SFB + BEZ inhibited the proliferation and cloning ability, blocked the cell cycle in the G0/G1 phase, and promoted apoptosis in the two cell lines. The autophagy level in Huh7R cells was higher than in Huh7 cells, and BEZ or SFB + BEZ further promoted autophagy in the two cell lines. In vivo, SFB + BEZ inhibited tumor growth by inducing apoptosis and autophagy. We concluded that BEZ235 enhanced the sensitivity of sorafenib through suppressing the PI3K/AKT/mTOR pathway and inducing autophagy. These observations may provide the experimental basis for sorafenib combined with BEZ235 in trial treatment of HCC.

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LRIG1 is a gatekeeper to exit from quiescence in adult neural stem cells

Nature Communications ◽

10.1038/s41467-021-22813-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

María Ángeles Marqués-Torrejón ◽

Charles A. C. Williams ◽

Benjamin Southgate ◽

Neza Alfazema ◽

Melanie P. Clements ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Neural Stem Cells ◽

Single Cell Analysis ◽

Interferon Response ◽

Quiescent State ◽

Adult Neural Stem Cells ◽

Protein Levels

AbstractAdult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence.

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

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3562-3576.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3562-3576 ◽

Cited By ~ 43

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00055-08 ◽

2008 ◽

Vol 28 (15) ◽

pp. 4653-4664 ◽

Cited By ~ 39

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.01358-09 ◽

2010 ◽

Vol 30 (16) ◽

pp. 3994-4005 ◽

Cited By ~ 26

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.

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The Cak1p Protein Kinase Is Required at G1/S and G2/M in the Budding Yeast Cell Cycle

Genetics ◽

10.1093/genetics/147.1.57 ◽

1997 ◽

Vol 147 (1) ◽

pp. 57-71 ◽

Cited By ~ 5

Author(s):

Ann Sutton ◽

Richard Freiman

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Cell Cycle Progression ◽

Budding Yeast ◽

Protein Kinase Activity ◽

Synthetic Lethal ◽

Protein Levels ◽

M Phase

Abstract The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G2 to M phase. Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G2 to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G1 into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function.

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

10.1101/2020.03.13.990127 ◽

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.

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