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

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

The Journal of Cell Biology ◽

10.1083/jcb.200104104 ◽

2001 ◽

Vol 154 (2) ◽

pp. 331-344 ◽

Cited By ~ 80

Author(s):

Daniel Kornitzer ◽

Rakefet Sharf ◽

Tamar Kleinberger

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Mammalian Cells ◽

Growth Arrest ◽

Anaphase Promoting Complex ◽

Reading Frame ◽

Cycle Growth ◽

M Phase ◽

Dependent Growth ◽

Synthetically Lethal

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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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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Involvement of calcineurin‐dependent degradation of Yap1p in Ca 2+ ‐induced G 2 cell‐cycle regulation in Saccharomyces cerevisiae

EMBO Reports ◽

10.1038/sj.embor.7400647 ◽

2006 ◽

Vol 7 (5) ◽

pp. 519-524 ◽

Cited By ~ 17

Author(s):

Hiroshi Yokoyama ◽

Masaki Mizunuma ◽

Michiyo Okamoto ◽

Josuke Yamamoto ◽

Dai Hirata ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cell Cycle Regulation ◽

Cycle Regulation

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Investigation of the cell cycle regulation of cdk3-associated kinase activity and the role of cdk3 in proliferation and transformation

Oncogene ◽

10.1038/sj.onc.1202145 ◽

1998 ◽

Vol 17 (17) ◽

pp. 2259-2269 ◽

Cited By ~ 21

Author(s):

Katja Braun ◽

Gabriele Hölzl ◽

Thomas Soucek ◽

Christoph Geisen ◽

Tarik Möröy ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Regulation ◽

Kinase Activity ◽

Cycle Regulation

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MAGE-C1/CT7 Inhibition Increases Myeloma Cell Line Sensitivity to Bortezomib-Induced Apoptosis: New Target for Myeloma Therapy?

Blood ◽

10.1182/blood.v116.21.1908.1908 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1908-1908

Author(s):

Fabricio de Carvalho ◽

Erico T. Costa ◽

Anamaria A. Camargo ◽

Juliana C. Gregorio ◽

Cibele Masotti ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Line ◽

Cell Cycle Regulation ◽

Myeloma Cell ◽

Myeloma Cell Line ◽

Empty Vector ◽

M Phase ◽

Induced Apoptosis ◽

Cycle Regulation

Abstract Abstract 1908 Introduction: MAGE-C1/CT7 encodes for a cancer/testis antigen (CTA) frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. The expression of this CTA is restricted to malignant plasma cells and a positive correlation between MAGEC1/CT7 expression and advanced stage has been demonstrated for MM. It has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function of this protein in the pathophysiology of MM is not yet understood. Objectives: (1) To clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle regulation in myeloma cell line SKO-007 and (2) to evaluate the impact of silencing MAGE-C1/CT7 on cells treated with bortezomib. Material and Methods: Short hairpin RNA (shRNA) specific for MAGE-C1/CT7 was inserted in the pRETROSUPER(pRS) retroviral vector. The pRS-shRNA-MAGE-C1/CT7 was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time PCR (RQ-PCR) and western blot. Functional studies included cell proliferation, cell cycle analysis using propidium iodide, and analysis of apoptosis using annexin V staining. Results: SKO-007 MM cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. SKO-007 cells were divided into three derivatives: empty vector (pRS) and ineffective shRNA (antisense strand deleted – GC bases) [both used as controls for all the experiments] and inhibited (shMAGE-C1/CT7). MAGE-C1/CT7 mRNA expression was ∼5 times lower in inhibited cell line than control cells by RQ-PCR. Western blot showed 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. We used empty vector, ineffective shRNA and inhibited cells to determine whether inhibition of MAGE-C1/CT7 was associated with cell cycle dysregulation. We detected differences between inhibited cells and both controls regarding the proportion of myeloma cells in the G2/M phase (p<0.05). When inhibited cells and controls were treated with 10 nM bortezomib for 48h, inhibited cells showed a 48% reduction of cells in the G2/M phase but control cells have 11% (empty vector) and 10% (ineffective shRNA) of reduction (p<0.05). Inhibited cells treated with 15 nM bortezomib showed an increased percentage of apoptotic cells in comparison with bortezomib treated controls (p<0.01) [Figure]. Conclusions: MAGE-C1/CT7 antigen inhibition did not change cell proliferation and DNA synthesis in SKO-007 cells. However, we found that MAGE-C1/CT7 plays in cell cycle regulation, protecting SKO-007 cells against bortezomib-induced apoptosis. Therefore, MAGE-C1/CT7 silencing by shRNA could be a strategy for future therapies in MM, i.e. in combination with proteasome inhibitors. [Supported by CNPq and LICR] Disclosures: No relevant conflicts of interest to declare.

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