scholarly journals Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition

2013 ◽  
Vol 203 (4) ◽  
pp. 595-604 ◽  
Author(s):  
Lenka Hálová ◽  
Wei Du ◽  
Sara Kirkham ◽  
Duncan L. Smith ◽  
Janni Petersen
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Binding Domain ◽  
Atp Binding ◽  
Dependent Manner ◽  
Tor Signaling ◽  
Cycle Arrest ◽  
Reduce Activity ◽  
Tor Kinase

TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase–controlled phosphorylation to generate physiologically significant changes in TOR signaling.

Vertebrate p34cdc2 phosphorylation site mutants: effects upon cell cycle progression in the fission yeast Schizosaccharomyces pombe

1992 ◽  
Vol 102 (1) ◽  
pp. 43-53 ◽  
Author(s):  
W. Krek ◽  
J. Marks ◽  
N. Schmitz ◽  
E.A. Nigg ◽  
V. Simanis
Keyword(s):  
Cell Cycle ◽  
Schizosaccharomyces Pombe ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Atp Binding ◽  
In Vitro Mutagenesis ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
P34 Cdc2

We have used the fission yeast Schizosaccharomyces pombe to analyse the effects of in vitro mutagenesis of the four known phosphorylation sites in the chicken p34(cdc2) protein, Thr 14, Tyr 15, Thr 161 and Ser 277, upon cell cycle progression. We have studied both the effect of overexpression of mutant proteins in a cdc2+ background and assayed their ability to rescue null and temperature-sensitive alleles of cdc2. Mutations of Thr 14 and Tyr 15 within the ATP binding domain of p34(cdc2) that mimic constitutive phosphorylation cause dominant negative cell cycle arrest when overexpressed. In contrast, some substitutions that simulate permanent dephosphorylation of the corresponding sites advance dephosphorylation of the corresponding sites advance mitosis. These data confirm the model that p34(cdc2) function is negatively regulated by phosphorylation of residues in the ATP binding site. Mutagenesis of the conserved residue Thr 161 functionally inactivates p34(cdc2), and our data suggest that both phosphorylation and dephosphorylation events at Thr 161 are required for progression through the cell cycle. Mutations at the fourth site of phosphorylation. Ser 277, lead to cold-sensitive cell cycle arrest, in minimal but not rich growth medium, suggesting that this site is involved in monitoring the nutritional status of the cell.

scholarly journals TORC2 dependent phosphorylation modulates calcium regulation of fission yeast myosin

2018 ◽  
Author(s):  
Karen Baker ◽  
Irene A. Gyamfi ◽  
Gregory I. Mashanov ◽  
Justin E. Molloy ◽  
Michael A. Geeves ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Fission Yeast ◽  
Single Molecule ◽  
Cell Cycle Progression ◽  
Neck Region ◽  
Dependent Manner ◽  
Meiotic Cell ◽  
Cycle Progression ◽  
Cellular Environment

AbstractAll cells have the ability to respond to changes in their environment. Signalling networks modulate cytoskeleton and membrane organisation to impact cell cycle progression, polarised cell growth and multicellular development according to the environmental setting. Using diverse in vitro, in vivo and single molecule techniques we have explored the role of myosin-1 signalling in regulating endocytosis during both mitotic and meiotic cell cycles. We have established that a conserved serine within the neck region of the sole fission yeast myosin-1 is phosphorylated in a TORC2 dependent manner to modulate myosin function. Myo1 neck phosphorylation brings about a change in the conformation of the neck region and modifies its interaction with calmodulins, Myo1 dynamics at endocytic foci, and promotes calcium dependent switching between different calmodulin light chains. These data provide insight into a novel mechanism by which myosin neck phosphorylation modulates acto-myosin dynamics to control polarised cell growth in response to mitotic and meiotic cell-cycle progression and the cellular environment.

scholarly journals Induction of cell cycle arrest via the p21, p27–cyclin E,A/Cdk2 pathway in SMMC-7721 hepatoma cells by clioquinol

2015 ◽  
Vol 65 (4) ◽  
pp. 463-471 ◽  
Author(s):  
Zhiwei Huang ◽  
Lianqiu Wang ◽  
Lifeng Chen ◽  
Yifei Zhang ◽  
Ping Shi
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Cycle Progression ◽  
Therapeutic Potential ◽  
Cyclin E ◽  
S Phase ◽  
Dependent Manner ◽  
Hepatic Cells ◽  
Cycle Arrest ◽  
First Time

Abstract Clioquinol has been shown to have anticancer activity in several carcinoma cells. In this study, we preliminarily examined the effect of clioquinol in human SMMC-7721 hepatoma and QSG-7701 normal hepatic cells. Our results indicated that clioquinol did not significantly affect survival of QSG-7701 cells, whereas it reduced cell viability in a concentration- and time-dependent manner in SMMC-7721 cells. Clioquinol did not trigger autophagy and apoptosis, while it induced cell cycle arrest in the S-phase in SMMC- 7721 cells. Additionally, down-regulation of cyclin D1, A2, E1, Cdk2 and up-regulation of p21, p27 were detected after the treatment with clioquinol. The results demonstrated for the first time that clioquinol suppressed cell cycle progression in the S-phase in SMMC-7721 cells via the p21, p27-cyclin E,A/Cdk2 pathway. This suggests that clioquinol may have a therapeutic potential as an anticancer drug for certain malignances.

scholarly journals Magnoflorine—Isolation and the Anticancer Potential against NCI-H1299 Lung, MDA-MB-468 Breast, T98G Glioma, and TE671 Rhabdomyosarcoma Cancer Cells

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1532
Author(s):  
Estera Okon ◽  
Wirginia Kukula-Koch ◽  
Marta Halasa ◽  
Agata Jarzab ◽  
Marzena Baran ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cancer Cells ◽  
Cell Cycle Progression ◽  
Dependent Manner ◽  
Current Standard ◽  
Aporphine Alkaloid ◽  
Cycle Arrest ◽  
Diphenyltetrazolium Bromide ◽  
Induction Of Apoptosis ◽  
High Doses

Magnoflorine (MGN) is a quaternary aporphine alkaloid that exhibits numerous therapeutic properties, including neuropsychopharmacological, anti-anxiety, immunomodulatory, anti-inflammatory, antioxidant, or antifungal activities. The aim of the present study was an investigation of the influence of MGN on viability, proliferation, induction of apoptosis, and cell cycle arrest in NCI-H1299 lung, MDA-MB-468 breast, T98G glioma, and TE671 rhabdomyosarcoma cancer cells. MGN was isolated from the roots of Berberis cretica L. by counter-current partition chromatography (CPC). Cell viability and proliferation assessments were performed by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and 5-bromo-2ʹ-deoxyuridine (BrDU) assays, respectively. The induction of apoptosis and cell cycle progression was measured using fluorescence-activated cell sorting analysis. MGN in high doses inhibits proliferation, induces apoptosis, and inhibits cell cycle in S/G2 phases in a dose-dependent manner. MGN seems to be a promising anti-cancer compound in therapy of some types of lung, breast, glioma, and rhabdomyosarcoma cancers, for which current standard therapies are limited or have severe strong side effects.

scholarly journals Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

1998 ◽  
Vol 9 (5) ◽  
pp. 1065-1080 ◽  
Author(s):  
Kenji Kitamura ◽  
Hiromi Maekawa ◽  
Chikashi Shimoda
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Restrictive Temperature ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Mitotic Cyclin

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role ofste9 + in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants ofste9 cdc10 ts, cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase.

scholarly journals An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation

2003 ◽  
Vol 160 (7) ◽  
pp. 1093-1103 ◽  
Author(s):  
Joseph J. Tasto ◽  
Jennifer L. Morrell ◽  
Kathleen L. Gould
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Fission Yeast ◽  
Cell Separation ◽  
Cell Cycle Progression ◽  
Dependent Manner ◽  
Septin Ring ◽  
Protein Levels ◽  
Division Site ◽  
Cell Biol

Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165–178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539–552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131–142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707–2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2Δ cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)–dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.

scholarly journals Murine Coronavirus Nonstructural Protein p28 Arrests Cell Cycle in G0/G1 Phase

2004 ◽  
Vol 78 (19) ◽  
pp. 10410-10419 ◽  
Author(s):  
Chun-Jen Chen ◽  
Kazuo Sugiyama ◽  
Hideyuki Kubo ◽  
Cheng Huang ◽  
Shinji Makino
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Cycle Progression ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Dependent Manner ◽  
Nonstructural Proteins ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Murine Coronavirus

ABSTRACT Murine coronavirus mouse hepatitis virus (MHV) gene 1 encodes several nonstructural proteins. The functions are unknown for most of these nonstructural proteins, including p28, which is encoded at the 5′ end of the MHV genome. Transient expression of cloned p28 in several different cultured cells inhibited cell growth, indicating that p28 expression suppressed cell proliferation. Expressed p28 was exclusively localized in the cytoplasm. Cell cycle analysis by flow cytometry demonstrated that p28 expression induced G0/G1 cell cycle arrest. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that p28 expression resulted in an accumulation of hypophosphorylated retinoblastoma protein (pRb), tumor suppressor p53, and cyclin-dependent kinase (Cdk) inhibitor p21Cip1. Expression of p28 did not alter the amount of p53 transcripts yet increased the amount of p21Cip1 transcripts, suggesting that p28 expression increased p53 stability and that p21Cip1 was transcriptionally activated in a p53-dependent manner. Our present data suggest the following model of p28-induced G0/G1 cell cycle arrest. Expressed cytoplasmic p28 induces the stabilization of p53, and accumulated p53 causes transcriptional upregulation of p21Cip1. The increased amount of p21Cip1 suppresses cyclin E/Cdk2 activity, resulting in the inhibition of pRb hyperphosphorylation. Accumulation of hypophosphorylated pRb thus prevents cell cycle progression from G0/G1 to S phase.

Pisosterol Induces G2/M Cell Cycle Arrest and Apoptosis via the ATM/ATR Signaling Pathway in Human Glioma Cells

2020 ◽  
Vol 20 (6) ◽  
pp. 734-750
Author(s):  
Wallax A.S. Ferreira ◽  
Rommel R. Burbano ◽  
Claudia do Ó. Pessoa ◽  
Maria L. Harada ◽  
Bárbara do Nascimento Borges ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Signaling Pathway ◽  
Glioma Cell ◽  
Molecular Mechanisms ◽  
Glioma Cells ◽  
Dependent Manner ◽  
M Cell ◽  
Trypan Blue Exclusion ◽  
Cycle Arrest

Background: Pisosterol, a triterpene derived from Pisolithus tinctorius, exhibits potential antitumor activity in various malignancies. However, the molecular mechanisms that mediate the pisosterol-specific effects on glioma cells remain unknown. Objective: This study aimed to evaluate the antitumoral effects of pisosterol on glioma cell lines. Methods: The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue exclusion assays were used to evaluate the effect of pisosterol on cell proliferation and viability in glioma cells. The effect of pisosterol on the distribution of the cells in the cell cycle was performed by flow cytometry. The expression and methylation pattern of the promoter region of MYC, ATM, BCL2, BMI1, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, MDM2, p14ARF and TP53 was analyzed by RT-qPCR, western blotting and bisulfite sequencing PCR (BSP-PCR). Results: Here, it has been reported that pisosterol markedly induced G2/M arrest and apoptosis and decreased the cell viability and proliferation potential of glioma cells in a dose-dependent manner by increasing the expression of ATM, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, p14ARF and TP53 and decreasing the expression of MYC, BCL2, BMI1 and MDM2. Pisosterol also triggered both caspase-independent and caspase-dependent apoptotic pathways by regulating the expression of Bcl-2 and activating caspase-3 and p53. Conclusions: It has been, for the first time, confirmed that the ATM/ATR signaling pathway is a critical mechanism for G2/M arrest in pisosterol-induced glioma cell cycle arrest and suggests that this compound might be a promising anticancer candidate for further investigation.

scholarly journals Slm9, a Novel Nuclear Protein Involved in Mitotic Control in Fission Yeast

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 623-631
Author(s):  
Junko Kanoh ◽  
Paul Russell
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Elongation ◽  
Nuclear Protein ◽  
G1 Arrest ◽  
Permissive Temperature ◽  
Cdc2 Kinase ◽  
Synthetic Lethal ◽  
Cycle Arrest ◽  
Novel Protein

Abstract In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis. Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1. Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2. To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25°). One of the genes, slm9, encodes a novel protein of 807 amino acids. Slm9 is most similar to Hir2, the histone gene regulator in budding yeast. Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle. The slm9 disruptant is delayed at the G2-M transition as indicated by cell elongation and analysis of DNA content. Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation. The slm9 mutant is defective in recovery from G1 arrest after nitrogen starvation. The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation.

scholarly journals A DNA-Damage-Induced Cell Cycle Checkpoint in Arabidopsis

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 323-334
Author(s):  
S B Preuss ◽  
A B Britt
Keyword(s):  
Cell Cycle ◽  
Gamma Radiation ◽  
Cell Cycle Progression ◽  
Cell Cycle Checkpoint ◽  
Phase Cell ◽  
Cycle Arrest ◽  
Cycle Checkpoint ◽  
Radiation Induced ◽  
High Doses ◽  
Regulation Of Cell Cycle

Abstract Although it is well established that plant seeds treated with high doses of gamma radiation arrest development as seedlings, the cause of this arrest is unknown. The uvh1 mutant of Arabidopsis is defective in a homolog of the human repair endonuclease XPF, and uvh1 mutants are sensitive to both the toxic effects of UV and the cytostatic effects of gamma radiation. Here we find that gamma irradiation of uvh1 plants specifically triggers a G2-phase cell cycle arrest. Mutants, termed suppressor of gamma (sog), that suppress this radiation-induced arrest and proceed through the cell cycle unimpeded were recovered in the uvh1 background; the resulting irradiated plants are genetically unstable. The sog mutations fall into two complementation groups. They are second-site suppressors of the uvh1 mutant's sensitivity to gamma radiation but do not affect the susceptibility of the plant to UV radiation. In addition to rendering the plants resistant to the growth inhibitory effects of gamma radiation, the sog1 mutation affects the proper development of the pollen tetrad, suggesting that SOG1 might also play a role in the regulation of cell cycle progression during meiosis.

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