Treatment of cells with the polyamine analog N
                        1
,N
                        11
-diethylnorspermine retards S phase progression within one cell cycle

Cyclins and CDKs (Cyclin Dependent Kinases) are key players in the biology of eukaryotic cells, representing hubs for the orchestration of physiological conditions with cell cycle progression. Furthermore, as in the case of meiosis, cyclins and CDKs have acquired novel functions unrelated to this primal role in driving the division cycle. Meiosis is a specialized developmental program that ensures proper propagation of the genetic information to the next generation by the production of gametes with accurate chromosome content, and meiosis-specific cyclins are widespread in evolution. We have explored the diversification of CDK functions studying the meiosis-specific Crs1 cyclin in fission yeast. In addition to the reported role in DSB (Double Strand Break) formation, this cyclin is required for meiotic S-phase progression, a canonical role, and to maintain the architecture of the meiotic chromosomes. Crs1 localizes at the SPB (Spindle Pole Body) and is required to stabilize the cluster of telomeres at this location (bouquet configuration), as well as for normal SPB motion. In addition, Crs1 exhibits CDK(Cdc2)-dependent kinase activity in a biphasic manner during meiosis, in contrast to a single wave of protein expression, suggesting a post-translational control of its activity. Thus, Crs1 displays multiple functions, acting both in cell cycle progression and in several key meiosis-specific events.

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CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

Biochemical Journal ◽

10.1042/bj3400135 ◽

1999 ◽

Vol 340 (1) ◽

pp. 135-141 ◽

Cited By ~ 23

Author(s):

Parisa DANAIE ◽

Michael ALTMANN ◽

Michael N. HALL ◽

Hans TRACHSEL ◽

Stephen B. HELLIWELL

Keyword(s):

Cell Cycle ◽

S Phase ◽

Translation Initiation Factor ◽

Yeast Cells ◽

Initiation Factor ◽

G1 Phase ◽

Mrna Levels ◽

Wild Type ◽

Phase Progression ◽

Type Gene

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

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Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

Molecular Biology of the Cell ◽

10.1091/mbc.e12-07-0548 ◽

2012 ◽

Vol 23 (21) ◽

pp. 4203-4211 ◽

Cited By ~ 9

Author(s):

Dong-Hwan Kim ◽

Deanna M. Koepp

Keyword(s):

Cell Cycle ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

S Phase ◽

Temperature Sensitive ◽

Dependent Manner ◽

Charged Residues ◽

Phase Progression ◽

Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

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EXTH-56. MEDULLOBLASTOMAS RESPOND TO CDK4/6 INHIBITION WITH NANOPARTICLE-DELIVERED PALBOCICLIB WITH ALTERED S-PHASE PROGRESSION

Neuro-Oncology ◽

10.1093/neuonc/noz175.387 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi94-vi94

Author(s):

Taylor Dismuke ◽

Chaemin Lim ◽

Timothy Gershon

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Flow Cytometric Analysis ◽

Pediatric Brain Tumors ◽

S Phase ◽

Flow Cytometric ◽

Phase Progression ◽

Reduced Rate ◽

G1 Cells ◽

Pediatric Brain

Abstract CDK4/6 inhibition is a promising therapy for medulloblastoma, one of the most common malignant pediatric brain tumors. To improve pharmacokinetics, we developed a polyoxazoline nanoparticle-encapsulated formulation of the FDA-approved CDK4/6 inhibitor palbociclib (POx-palbo). We then administered POx-palbo to transgenic medulloblastoma-prone GFAP-Cre/SmoM2 mice, to determine the efficacy and mechanisms of action and resistance. We found that POx-palbo slowed tumor progression, but consistently failed to be curative. Further analysis showed that while CDK4/6 inhibition acutely blocked G1 cells from re-entering the cell cycle, this effect wore off within hours of drug administration. However, flow cytometric analysis of EdU uptake hours after palbociclib demonstrated aberrant S-phase with reduced rate of DNA synthesis. This POx-palbociclib-induced alteration of S-phase progression seems to remain true at later time points even when we observed that palbociclib G1/S inhibition began to decrease. Based on these data, we propose that the combinational therapy of POx-palbociclib and S-phase targeting agents will further improve treatment. Faulty tumor cell cycle progression in the presence of Pox-palbociclib may give increased window to target the S-phase for irreversible cell-cycle exit.

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Novel Control of S Phase of the Cell Cycle by Ubiquitin-conjugating Enzyme H7

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0036 ◽

2009 ◽

Vol 20 (1) ◽

pp. 1-9 ◽

Cited By ~ 19

Author(s):

Elizabeth A. Whitcomb ◽

Edward J. Dudek ◽

Qing Liu ◽

Allen Taylor

Keyword(s):

Cell Cycle ◽

S Phase ◽

Regulatory Proteins ◽

Protein Levels ◽

Proteolytic Pathway ◽

Dynamic Cell ◽

Ubiquitin Conjugating Enzyme ◽

Phase Progression ◽

Cycle Dependent ◽

Cycle Regulation

Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle–dependent changes in levels of UbcH7 regulate entrance into and progression through S phase. In diverse cell lines, UbcH7 protein levels are dramatically reduced in S phase but are fully restored by G2. Knockdown of UbcH7 increases the proportion of cells in S phase and doubles the time to traverse S phase, whereas UbcH7 overexpression reduces the proportion of cells in S phase. These data suggest a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, UbcH7 knockdown was coincident with elevated levels of the checkpoint kinase Chk1 but not Chk2. These results argue that UbcH7 promotes S phase progression to G2 by modulating the intra-S phase checkpoint mediated by Chk1. Furthermore, UbcH7 levels appear to be regulated by a UPP. Together the data identify novel roles for the UPP, specifically UbcH7 in the regulation of S phase transit time as well as in cell proliferation.

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Cyclin A2/Cdk1 is Essential for the in vivo S Phase Entry by Phosphorylating Top2a

10.1101/513861 ◽

2019 ◽

Author(s):

Miaomiao Jin ◽

Ruikun Hu ◽

Baijie Xu ◽

Weilai Huang ◽

Hong Wang ◽

...

Keyword(s):

Cell Cycle ◽

Cyclin B1 ◽

S Phase ◽

Downstream Target ◽

Early Embryonic Lethality ◽

M Phase ◽

Phase Progression ◽

Cyclin A2 ◽

Phase Entry

AbstractCyclin-dependent kinase 1 (CDK1) plays essential roles in cell cycle regulation. However, due to the early embryonic lethality of mouse Cdk1 mutants, the in vivo role of CDK1 in regulating cell cycle and embryonic development remains unclear. Here, by generating zebrafish cdk1 mutants using CRISPR/Cas9 system, we show that cdk1−/− embryos exhibit severe microphthalmia accompanied with multiple defects in polarized cell division, S phase entry and M phase progression, cell apoptosis and cell differentiation, but not in interkinetic nuclear migration (IKNM). By informatics analysis, we identified Top2a as a potential downstream target, and Cyclin A2 and Cyclin B1 as partners of Cdk1 in cell cycle. Depletion of either Cyclin A2 or Top2a leads to decreased S phase entry and increased DNA damage response in zebrafish retinal cells, and depletion of Cyclin B1 leads to M phase arrest. Immunoprecipitation shows that Cdk1 and Cyclin A2 physically interact in vivo. Moreover, phosphorylation of Top2a on Serine 1213 (S1213) site is almost absent in either cdk1 or ccna2 mutants, but in not ccnb1 mutants. Furthermore, overexpression of TOP2AS1213, the phosphomimetic form of human TOP2A, rescues S phase entry and microphthalmia defects in cdk1−/− and ccna2−/− embryos. Taken together, our data suggests that Cdk1 interacts with Cyclin A2 to regulate S phase entry through phosphorylating Top2a, and with Cyclin B1 to regulate M phase progression in vivo.

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p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability

Nature Communications ◽

10.1038/s41467-020-18838-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Longtao Wu ◽

Clayton D. Crawley ◽

Andrea Garofalo ◽

Jackie W. Nichols ◽

Paige-Ashley Campbell ◽

...

Keyword(s):

Cell Cycle ◽

Genome Stability ◽

Cell Cycle Progression ◽

Human Cancer ◽

S Phase ◽

Post Translational Modification ◽

Chromosomal Breakage ◽

Cycle Progression ◽

Genome Wide ◽

Phase Progression

Abstract p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.

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Faculty Opinions recommendation of MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1023391.280684 ◽

2005 ◽

Author(s):

Jane Endicott

Keyword(s):

Cell Cycle ◽

Uv Irradiation ◽

S Phase ◽

Cell Cycle Checkpoint ◽

Checkpoint Kinase ◽

Cycle Checkpoint ◽

A Cell ◽

Phase Progression

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ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Blood ◽

10.1182/blood.v128.22.5624.5624 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5624-5624

Author(s):

Dhyani Anamika ◽

Patricia Favaro ◽

Sara Teresinha Olalla Saad

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Dna Damage ◽

S Phase ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Myeloma Cells ◽

Checkpoint Kinase ◽

Strand Breaks ◽

Phase Progression

Abstract Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells. In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012; 586: 4311-18. 2) ANKHD1 is essential for repair of DNA double strand breaks in multiple myeloma. Dhyani et al. ASH Abstract, Blood 2015; 126:1762. Disclosures No relevant conflicts of interest to declare.

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Cooperative Regulation of the Cell Division Cycle by the Protein Kinases RAF and AKT

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10868-10881.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10868-10881 ◽

Cited By ~ 78

Author(s):

Amer M. Mirza ◽

Stephan Gysin ◽

Nisar Malek ◽

Kei-ichi Nakayama ◽

James M. Roberts ◽

...

Keyword(s):

Cell Cycle ◽

Cancer Cells ◽

Signaling Pathways ◽

Protein Kinases ◽

Akt Signaling ◽

S Phase ◽

Pi3 Kinase ◽

Human Pancreatic Cancer ◽

Phase Cell ◽

Phase Progression

ABSTRACT The RAS-activated RAF→MEK→extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′-kinase (PI3′-kinase)→PDK1→AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G1 cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27Kip1 expression. Repression of p27Kip1 was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27Kip1. Consistent with these observations, pharmacological inhibition of MEK or PI3′-kinase inhibited the effects of activated RAS on the expression of p27Kip1 in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21Cip1 from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF→MEK→ERK and PI3′K→PDK→AKT signaling pathways can cooperate to promote G0→G1→S-phase cell cycle progression in both normal and cancer cells.

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