Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)–dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4–PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.

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Cell Cycle Regulation of the Saccharomyces cerevisiae Polo-Like Kinase Cdc5p

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7360 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7360-7370 ◽

Cited By ~ 71

Author(s):

Liang Cheng ◽

Linda Hunke ◽

Christopher F. J. Hardy

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cell Cycle Regulation ◽

Kinase Activity ◽

Anaphase Promoting Complex ◽

Mitotic Kinases ◽

Polo Kinase ◽

Evolutionarily Conserved ◽

M Phase ◽

Cycle Regulation

ABSTRACT Progression through and completion of mitosis require the actions of the evolutionarily conserved Polo kinase. We have determined that the levels of Cdc5p, a Saccharomyces cerevisiae member of the Polo family of mitotic kinases, are cell cycle regulated. Cdc5p accumulates in the nuclei of G2/M-phase cells, and its levels decline dramatically as cells progress through anaphase and begin telophase. We report that Cdc5p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). We have determined that Cdc5p-associated kinase activity is restricted to G2/M and that this activity is posttranslationally regulated. These results further link the actions of the APC to the completion of mitosis and suggest possible roles for Cdc5p during progression through and completion of mitosis.

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Cell Cycle-Dependent Expression of Mammalian E2-C Regulated by the Anaphase-Promoting Complex/Cyclosome

Molecular Biology of the Cell ◽

10.1091/mbc.11.8.2821 ◽

2000 ◽

Vol 11 (8) ◽

pp. 2821-2831 ◽

Cited By ~ 36

Author(s):

Atsushi Yamanaka ◽

Shigetsugu Hatakeyama ◽

Kin-ichiro Kominami ◽

Masatoshi Kitagawa ◽

Masaki Matsumoto ◽

...

Keyword(s):

Cell Cycle ◽

Substrate Specificity ◽

Critical Role ◽

Anaphase Promoting Complex ◽

Polyubiquitin Chain ◽

M Phase ◽

Ubiquitin Conjugating Enzyme ◽

Recognition Motifs ◽

Cycle Dependent ◽

Conjugating Enzyme

Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Here we report that mammalian E2-C is expressed in late G2/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity.

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Cooperative effects of genes controlling the G2/M checkpoint

Genes & Development ◽

10.1101/gad.14.13.1584 ◽

2000 ◽

Vol 14 (13) ◽

pp. 1584-1588

Author(s):

Timothy A. Chan ◽

Paul M. Hwang ◽

Heiko Hermeking ◽

Kenneth W. Kinzler ◽

Bert Vogelstein

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Mammalian Cells ◽

Growth Arrest ◽

Double Knockout ◽

Specific Test ◽

Cooperative Effects ◽

Exogenous Expression ◽

Induced Genes ◽

Single Knockout

It is believed that multiple effectors independently control the checkpoints permitting transitions between cell cycle phases. However, this has not been rigorously demonstrated in mammalian cells. The p53-induced genes p21 and 14-3-3ς are each required for the G2 arrest and allow a specific test of this fundamental tenet. We generated human cells deficient in bothp21 and 14-3-3ς and determined whether the double knockout was more sensitive to DNA damage than either single knockout.p21−/−14-3-3ς−/− cells were significantly more sensitive to DNA damage or to the exogenous expression of p53 than cells lacking only p21 or only 14-3-3ς. Thus, p21 and 14-3-3ς play distinct but complementary roles in the G2/M checkpoint, and help explain why genes at the nodal points of growth arrest pathways, like p53, are the targets of mutation in cancer cells.

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Posttranslational Phosphorylation and Ubiquitination of the Saccharomyces cerevisiae Poly(A) Polymerase at the S/G2 Stage of the Cell Cycle

Molecular and Cellular Biology ◽

10.1128/mcb.20.8.2794-2802.2000 ◽

2000 ◽

Vol 20 (8) ◽

pp. 2794-2802 ◽

Cited By ~ 13

Author(s):

Neptune Mizrahi ◽

Claire Moore

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cell Sorting ◽

S Phase ◽

Phase Arrest ◽

Phosphatase Treatment ◽

M Phase ◽

64 Kda Protein ◽

Mitotic Phosphorylation ◽

Cycle Regulation

ABSTRACT The poly(A) polymerase of the budding yeast Saccharomyces cerevisiae (Pap1) is a 64-kDa protein essential for the maturation of mRNA. We have found that a modified Pap1 of 90 kDa transiently appears in cells after release from α-factor-induced G1 arrest or from a hydroxyurea-induced S-phase arrest. While a small amount of modification occurs in hydroxyurea-arrested cells, fluorescence-activated cell sorting analysis and microscopic examination of bud formation indicate that the majority of modified enzyme is found at late S/G2 and disappears by the time cells have reached M phase. The reduction of the 90-kDa product upon phosphatase treatment indicates that the altered mobility is due to phosphorylation. A preparation containing primarily the phosphorylated Pap1 has no poly(A) addition activity, but this activity is restored by phosphatase treatment. A portion of Pap1 is also polyubiquitinated concurrent with phosphorylation. However, the bulk of the 64-kDa Pap1 is a stable protein with a half-life of 14 h. The timing, nature, and extent of Pap1 modification in comparison to the mitotic phosphorylation of mammalian poly(A) polymerase suggest an intriguing difference in the cell cycle regulation of this enzyme in yeast and mammalian systems.

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Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a Chemical Genetics Approach

10.26686/wgtn.16969795.v1 ◽

2021 ◽

Author(s):

◽

Reem Hanna

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Amino Acids ◽

Flow Cytometry ◽

Binding Site ◽

Mammalian Cells ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Peloruside A ◽

Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

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Identification of Novel Human Cdt1-binding Proteins by a Proteomics Approach: Proteolytic Regulation by APC/CCdh1

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0859 ◽

2008 ◽

Vol 19 (3) ◽

pp. 1007-1021 ◽

Cited By ~ 44

Author(s):

Nozomi Sugimoto ◽

Issay Kitabayashi ◽

Satoko Osano ◽

Yasutoshi Tatsumi ◽

Takashi Yugawa ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Mammalian Cells ◽

Topoisomerase I ◽

Binding Proteins ◽

Mass Spectrometry Analysis ◽

Anaphase Promoting Complex ◽

Chromosomal Damage ◽

Spectrometry Analysis

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIα, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/CCdh1, SNF2H, topoisomerase I and IIα, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/CCdh1 indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCFSkp2 and cullin4-based ubiquitin ligases, APC/CCdh1 is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.

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Cell Cycle Regulation of DNA Replication Initiator Factor Dbf4p

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4270 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4270-4278 ◽

Cited By ~ 54

Author(s):

Liang Cheng ◽

Tim Collyer ◽

Christopher F. J. Hardy

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Genetic Material ◽

Anaphase Promoting Complex ◽

Initiator Protein ◽

Evolutionarily Conserved ◽

M Phase ◽

Cycle Regulation ◽

Replication Initiator

ABSTRACT The precise duplication of eukaryotic genetic material takes place once and only once per cell cycle and is dependent on the completion of the previous mitosis. Two evolutionarily conserved kinases, the cyclin B (Clb)/cyclin-dependent kinase (Cdk/Cdc28p) and Cdc7p along with its interacting factor Dbf4p, are required late in G1 to initiate DNA replication. We have determined that the levels of Dbf4p are cell cycle regulated. Dbf4p levels increase as cells begin S phase and remain high through late mitosis, after which they decline dramatically as cells begin the next cell cycle. We report that Dbf4p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). In addition, Dbf4p is modified in response to DNA damage, and this modification is dependent upon the DNA damage response pathway. We had previously shown that Dbf4p interacts with the M phase polo-like kinase Cdc5p, a key regulator of the APC late in mitosis. These results further link the actions of the initiator protein, Dbf4p, to the completion of mitosis and suggest possible roles for Dbf4p during progression through mitosis.

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Coupling Phosphate Homeostasis to Cell Cycle-Specific Transcription: Mitotic Activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.00222-09 ◽

2009 ◽

Vol 29 (18) ◽

pp. 4891-4905 ◽

Cited By ~ 16

Author(s):

Santhi Pondugula ◽

Daniel W. Neef ◽

Warren P. Voth ◽

Russell P. Darst ◽

Archana Dhasarathy ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cell Cycle Progression ◽

Mitotic Cell ◽

Dependent Manner ◽

Phosphate Homeostasis ◽

Periodic Cell ◽

M Phase ◽

Cycle Dependent ◽

Nutrient Homeostasis

ABSTRACT Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G2/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G1. These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G1 induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

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Synergistic inhibition of APC/C by glucose and activated Ras proteins can be mediated by each of the Tpk1–3 proteins in Saccharomyces cerevisiae

Microbiology ◽

10.1099/mic.0.26062-0 ◽

2003 ◽

Vol 149 (5) ◽

pp. 1205-1216 ◽

Cited By ~ 19

Author(s):

Melanie Bolte ◽

Patrick Dieckhoff ◽

Cindy Krause ◽

Gerhard H. Braus ◽

Stefan Irniger

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Negative Regulator ◽

Anaphase Promoting Complex ◽

Ras Proteins ◽

Dependent Protein Kinase ◽

Glucose Addition ◽

Dependent Protein ◽

Pka Pathway ◽

Redundant Functions

Proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for the progression through mitosis. APC/C is a highly conserved ubiquitin ligase whose activity is regulated during the cell cycle by various factors, including spindle checkpoint components and protein kinases. The cAMP-dependent protein kinase (PKA) was identified as negative regulator of APC/C in yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, PKA activity is induced upon glucose addition or by activated Ras proteins. This study shows that glucose and the activated Ras2Val19 protein synergistically inhibit APC/C function via the cAMP/PKA pathway in yeast. Remarkably, Ras2 proteins defective in the interaction with adenylate cyclase fail to influence APC/C, implying that its function is regulated exclusively by PKA, but not by alternative Ras pathways. Furthermore, it is shown that the three PKAs in yeast, Tpk1, Tpk2 and Tpk3, have redundant functions in regulating APC/C in response to glucose medium. Single or double deletions of TPK genes did not prevent inhibition of APC/C, suggesting that each of the Tpk proteins can take over this function. However, Tpk2 seems to inhibit APC/C function more efficiently than Tpk1 and Tpk3. Finally, evidence is provided that Cdc20 is involved in APC/C regulation by the cAMP/PKA pathway.

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