scholarly journals Phosphorylation by Casein Kinase 2 Regulates Nap1 Localization and Function

2007 ◽  
Vol 28 (4) ◽  
pp. 1313-1325 ◽  
Author(s):  
Meredith E. K. Calvert ◽  
Kristin M. Keck ◽  
Celeste Ptak ◽  
Jeffrey Shabanowitz ◽  
Donald F. Hunt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
S Phase ◽  
Bud Formation ◽  
Evolutionarily Conserved ◽  
Assembly Factor ◽  
And Function ◽  
Cycle Regulation

ABSTRACT In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.

scholarly journals Cyclin F Disruption Compromises Placental Development and Affects Normal Cell Cycle Execution

2004 ◽  
Vol 24 (6) ◽  
pp. 2487-2498 ◽  
Author(s):  
Michael T. Tetzlaff ◽  
Chang Bai ◽  
Milton Finegold ◽  
John Wilson ◽  
J. Wade Harper ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Placental Development ◽  
Cell Cycle Reentry ◽  
High Conservation ◽  
And Function ◽  
Cyclin F ◽  
Cycle Regulation

ABSTRACT Human cyclin F was originally isolated as a cDNA capable of suppressing the temperature sensitivity of a Saccharomyces cerevisiae cdc4-1 mutant. Its tightly regulated expression and high conservation in the evolutionary progression from amphibians to mammals suggest that it coordinates the timing of a critical cell cycle event. The fact that it contains an F box and can form an SCF (Skp1-Cul1/Cdc53-F-box) complex in vivo further suggests that it may also function in proteolysis. To investigate the role of cyclin F in vivo, we generated mice deficient for cyclin F and conditionally deficient mice as well as mouse embryonic fibroblasts (MEFs) conditionally deficient for cyclin F. Heterozygous animals are normal and fertile, but CycF−/− animals, with a myriad of developmental anomalies due in large part to failures in yolk sac and chorioallantoic placentation, die around embryonic day 10.5. Tissue-specific deletion of cyclin F revealed that it was not required for the development and function of a number of different embryonic and adult tissues. In contrast, MEFs lacking cyclin F, while viable, do exhibit cell cycle defects, including reduced population-doubling time and a delay in cell cycle reentry from quiescence, indicating that cyclin F plays a role in cell cycle regulation.

scholarly journals DNA Damage-Induced Cell Cycle Regulation and Function of Novel Chk2 Phosphoresidues

2006 ◽  
Vol 26 (21) ◽  
pp. 7832-7845 ◽  
Author(s):  
Giacomo Buscemi ◽  
Luigi Carlessi ◽  
Laura Zannini ◽  
Sofia Lisanti ◽  
Enrico Fontanella ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Dna Double Strand Breaks ◽  
Independent Manner ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
And Function ◽  
Cycle Regulation

ABSTRACT Chk2 kinase is activated by DNA damage to regulate cell cycle arrest, DNA repair, and apoptosis. Phosphorylation of Chk2 in vivo by ataxia telangiectasia-mutated (ATM) on threonine 68 (T68) initiates a phosphorylation cascade that promotes the full activity of Chk2. We identified three serine residues (S19, S33, and S35) on Chk2 that became phosphorylated in vivo rapidly and exclusively in response to ionizing radiation (IR)-induced DNA double-strand breaks in an ATM- and Nbs1-dependent but ataxia telangiectasia- and Rad3-related-independent manner. Phosphorylation of these residues, restricted to the G1 phase of the cell cycle, was induced by a higher dose of IR (>1 Gy) than that required for phosphorylation of T68 (0.25 Gy) and declined by 45 to 90 min, concomitant with a rise in Chk2 autophosphorylation. Compared to the wild-type form, Chk2 with alanine substitutions at S19, S33, and S35 (Chk2S3A) showed impaired dimerization, defective auto- and trans-phosphorylation activities, and reduced ability to promote degradation of Hdmx, a phosphorylation target of Chk2 and regulator of p53 activity. Besides, Chk2S3A failed to inhibit cell growth and, in response to IR, to arrest G1/S progression. These findings underscore the critical roles of S19, S33, and S35 and argue that these phosphoresidues may serve to fine-tune the ATM-dependent response of Chk2 to increasing amounts of DNA damage.

Novel Anthraquinone Derivatives as Dual Inhibitors of Topoisomerase 2 and Casein Kinase 2: In Silico Studies, Synthesis and Biological Evaluation on Leukemic Cell Lines

2019 ◽  
Vol 18 (11) ◽  
pp. 1551-1562 ◽  
Author(s):  
Abbas Kabir ◽  
Kalpana Tilekar ◽  
Neha Upadhyay ◽  
C.S. Ramaa
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Biological Evaluation ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Cell Cycle Analysis ◽  
Protein Targets ◽  
Topoisomerase 2 ◽  
Dual Inhibitors

Background: Cancer being a complex disease, single targeting agents remain unsuccessful. This calls for “multiple targeting”, wherein a single drug is so designed that it will modulate the activity of multiple protein targets. Topoisomerase 2 (Top2) helps in removing DNA tangles and super-coiling during cellular replication, Casein Kinase 2 (CK2) is involved in the phosphorylation of a multitude of protein targets. Thus, in the present work, we have tried to develop dual inhibitors of Top2 and CK2. Objective: With this view, in the present work, 2 human proteins, Top2 and CK2 have been targeted to achieve the anti-proliferative effects. Methods: Novel 1-acetylamidoanthraquinone (3a-3y) derivatives were designed, synthesized and their structures were elucidated by analytical and spectral characterization techniques (FTIR, 1H NMR, 13C NMR and Mass Spectroscopy). The synthesized compounds were then subjected to evaluation of cytotoxic potential by the Sulforhodamine B (SRB) protein assay, using HL60 and K562 cell lines. Ten compounds were analyzed for Top2, CK2 enzyme inhibitory potential. Further, top three compounds were subjected to cell cycle analysis. Results: The compounds 3a to 3c, 3e, 3f, 3i to 3p, 3t and 3x showed excellent cytotoxic activity to HL-60 cell line indicating their high anti-proliferative potential in AML. The compounds 3a to 3c, 3e, 3f, 3i to 3p and 3y have shown good to moderate activity on K-562 cell line. Compounds 3e, 3f, 3i, 3x and 3y were found more cytotoxic than standard doxorubicin. In cell cycle analysis, the cells (79-85%) were found to arrest in the G0/G1 phase. Conclusion: We have successfully designed, synthesized, purified and structurally characterized 1- acetylamidoanthraquinone derivatives. Even though our compounds need design optimization to further increase enzyme inhibition, their overall anti-proliferative effects were found to be encouraging.

scholarly journals Spanning the gap: unraveling RSC dynamics in vivo

2021 ◽  
Author(s):  
Heinz Neumann ◽  
Bryan J. Wilkins
Keyword(s):  
Cell Cycle ◽  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Binding Mode ◽  
Binding Modes ◽  
Binding Domains ◽  
Binding Interface ◽  
The Many ◽  
And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

Cdc6p establishes and maintains a state of replication competence during G1 phase

1997 ◽  
Vol 110 (6) ◽  
pp. 753-763 ◽  
Author(s):  
C.S. Detweiler ◽  
J.J. Li
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Transcriptional Repression ◽  
Prolonged Exposure ◽  
S Phase ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Important Intermediate ◽  
Initiation Of Dna Replication ◽  
And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

scholarly journals Human Enhancer of Invasion-Cluster, a Coiled-Coil Protein Required for Passage through Mitosis

2004 ◽  
Vol 24 (9) ◽  
pp. 3957-3971 ◽  
Author(s):  
Margret B. Einarson ◽  
Edna Cukierman ◽  
Duane A. Compton ◽  
Erica A. Golemis
Keyword(s):  
Cell Cycle ◽  
Coiled Coil ◽  
Cell Cycle Checkpoints ◽  
Mitotic Spindles ◽  
Human Genes ◽  
Hydroxyurea Treatment ◽  
Evolutionarily Conserved ◽  
Defects Analysis ◽  
Spindle Function

ABSTRACT In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G2/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.

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

2000 ◽  
Vol 20 (8) ◽  
pp. 2794-2802 ◽  
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.

Cell Cycle Regulation of E2F Site Occupation in Vivo

Science ◽  
1996 ◽  
Vol 271 (5255) ◽  
pp. 1595-1597 ◽  
Author(s):  
J. Zwicker ◽  
N. Liu ◽  
K. Engeland ◽  
F. C. Lucibello ◽  
R. Muller
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Site Occupation ◽  
Cycle Regulation

scholarly journals Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1647-1653 ◽  
Author(s):  
A Raza ◽  
Y Maheshwari ◽  
HD Preisler
Keyword(s):  
Cell Cycle ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Total Cell ◽  
Acute Nonlymphocytic Leukemia ◽  
Cell Cycle Time ◽  
Myeloid Leukemias ◽  
History Of

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post- BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

scholarly journals Alteration of the proliferative rate of acute myelogenous leukemia cells in vivo in patients

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2600-2603 ◽  
Author(s):  
HD Preisler ◽  
A Raza ◽  
RA Larson
Keyword(s):  
Cell Cycle ◽  
Acute Myelogenous Leukemia ◽  
S Phase ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Cell Cycle Time ◽  
Proliferative Rate ◽  
Acute Myelogenous ◽  
Bioactive Agents

Abstract Ten patients with active acute myelogenous leukemia (AML) received either 13 cis retinoic acid (RA) + alpha interferon (IFN) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 3 days. Cell cycle measurements were performed before and at the conclusion of administration of the bioactive agent(s). The proliferative rate of the leukemia cells in vivo decreased in four of five patients receiving RA+IFN whereas in one patient proliferation accelerated. The proliferative rate of AML cells accelerated in three of the five patients who received rhGM-CSF and slowed in two patients. These data show that while the proliferative rate of AML cells can be altered in vivo, the effect produced by bioactive agents may be the opposite of the desired effect. Furthermore, the studies described here demonstrate the usefulness of marrow biopsies for measuring the percent S-phase cells and the importance of measuring the duration of S phase so that the effects of bioactive agents on the cell cycle time of the leukemia cells can be determined.

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