scholarly journals Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage

1998 ◽  
Vol 141 (4) ◽  
pp. 875-885 ◽  
Author(s):  
Pei Jin ◽  
Stephen Hardy ◽  
David O. Morgan
Keyword(s):  
Dna Damage ◽  
Normal Growth ◽  
Subcellular Location ◽  
Cyclin B1 ◽  
Growth Conditions ◽  
Human Cells ◽  
Entry And Exit ◽  
Nuclear Targeting ◽  
G2 Arrest ◽  
Mitotic Entry

Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.

scholarly journals Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells.

1996 ◽  
Vol 134 (4) ◽  
pp. 963-970 ◽  
Author(s):  
P Jin ◽  
Y Gu ◽  
D O Morgan
Keyword(s):  
Dna Damage ◽  
Chromatin Condensation ◽  
S Phase ◽  
Human Cells ◽  
G2 Arrest ◽  
Hela Cell Lines ◽  
Radiation Induced ◽  
G2 Delay ◽  
In Cells

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.

scholarly journals p21-Mediated Nuclear Retention of Cyclin B1-Cdk1 in Response to Genotoxic Stress

2004 ◽  
Vol 15 (9) ◽  
pp. 3965-3976 ◽  
Author(s):  
Fabienne Baus Charrier-Savournin ◽  
Marie-Thérèse Château ◽  
Véronique Gire ◽  
John Sedivy ◽  
Jacques Piette ◽  
...  
Keyword(s):  
Dna Damage ◽  
Current Model ◽  
Human Fibroblasts ◽  
Genotoxic Stress ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
G2 Arrest ◽  
Nuclear Retention ◽  
Mitotic Cyclin ◽  
Normal Human

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.

scholarly journals Repression of CDK1 and Other Genes with CDE and CHR Promoter Elements during DNA Damage-Induced G2/M Arrest in Human Cells

2000 ◽  
Vol 20 (7) ◽  
pp. 2358-2366 ◽  
Author(s):  
Christophe Badie ◽  
Jane E. Itzhaki ◽  
Matthew J. Sullivan ◽  
Adam J. Carpenter ◽  
Andrew C. G. Porter
Keyword(s):  
Dna Damage ◽  
Transcriptional Repression ◽  
Human Fibroblasts ◽  
Cyclin B1 ◽  
Human Cells ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Cyclin Dependent Kinase ◽  
Promoter Regions ◽  
Topoisomerase Iiα

ABSTRACT Entry into mitosis is controlled by the cyclin-dependent kinase CDK1 and can be delayed in response to DNA damage. In some systems, such G2/M arrest has been shown to reflect the stabilization of inhibitory phosphorylation sites on CDK1. In human cells, full G2 arrest appears to involve additional mechanisms. We describe here the prolonged (>6 day) downregulation of CDK1 protein and mRNA levels following DNA damage in human cells. This silencing of gene expression is observed in primary human fibroblasts and in two cell lines with functional p53 but not in HeLa cells, where p53 is inactive. Silencing is accompanied by the accumulation of cells in G2, when CDK1 expression is normally maximal. The response is impaired by mutations in cis-acting elements (CDE and CHR) in the CDK1 promoter, indicating that silencing occurs at the transcriptional level. These elements have previously been implicated in the repression of transcription during G1that is normally lifted as cells progress into S and G2. Interestingly, we find that other genes, including those for CDC25C, cyclin A2, cyclin B1, CENP-A, and topoisomerase IIα, that are normally expressed preferentially in G2 and whose promoter regions include putative CDE and CHR elements are also downregulated in response to DNA damage. These data, together with those of other groups, support the existence of a p53-dependent, DNA damage-activated pathway leading to CHR- and CDE-mediated transcriptional repression of various G2-specific genes. This pathway may be required for sustained periods of G2 arrest following DNA damage.

scholarly journals TIP49b, a Regulator of Activating Transcription Factor 2 Response to Stress and DNA Damage

2001 ◽  
Vol 21 (24) ◽  
pp. 8398-8413 ◽  
Author(s):  
Ssang-Goo Cho ◽  
Anindita Bhoumik ◽  
Limor Broday ◽  
Vladimir Ivanov ◽  
Barry Rosenstein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Transcription Factor ◽  
Transcriptional Control ◽  
Constitutive Expression ◽  
Normal Growth ◽  
Growth Conditions ◽  
Interacting Protein ◽  
Response To Stress ◽  
Activating Transcription Factor

ABSTRACT Activating transcription factor 2 (ATF2/CRE-BP1) is implicated in transcriptional control of stress-responsive genes. A yeast two-hybrid screen identified TBP-interacting protein 49b (TIP49b), a component of the INO80 chromatin-remodeling complex, as a novel ATF2-interacting protein. TIP49b's association with ATF2 is phosphorylation dependent and requires amino acids 150 to 248 of ATF2 (ATF2150–248), which are implicated in intramolecular inhibition of ATF2 transcriptional activities. Forced expression of TIP49b efficiently attenuated ATF2 transcriptional activities under normal growth conditions as well as after UV treatment, ionizing irradiation, or activation of p38 kinase, all of which induced ATF2 phosphorylation and increased TIP49b-ATF2 association. Constitutive expression of ATF2150–248 peptide outcompeted TIP49b interaction with ATF2 and alleviated the suppression of ATF2 transcriptional activities. Expression of ATF2150–248 in fibroblasts or melanoma but not in ATF2-null cells caused a profound G2M arrest and increased degree of apoptosis following irradiation. The interaction between ATF2 and TIP49b constitutes a novel mechanism that serves to limit ATF2 transcriptional activities and highlights the central role of ATF2 in the control of the cell cycle and apoptosis in response to stress and DNA damage.

scholarly journals Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Amrutha Kizhedathu ◽  
Archit V Bagul ◽  
Arjun Guha
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Negative Regulation ◽  
Dependent Manner ◽  
G2 Arrest ◽  
Organ Growth ◽  
Checkpoint Kinase ◽  
Mitotic Entry ◽  
Checkpoint Kinase 1 ◽  
Larval Stages

Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. Here we investigate the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). We report that tracheoblasts pause in G2 for ~48–56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). We find that mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24–32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. We propose that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.

scholarly journals Entry to and exit from diapause arrest in Caenorhabditis elegans are both regulated by a steroid hormone pathway

2021 ◽  
Author(s):  
Mark G Zhang ◽  
Paul W Sternberg
Keyword(s):  
Caenorhabditis Elegans ◽  
Hormonal Regulation ◽  
Steroid Hormone ◽  
Normal Growth ◽  
Growth Conditions ◽  
Hormone Signaling ◽  
Hormone Regulation ◽  
Entry And Exit ◽  
Steroid Hormone Signaling ◽  
Summary Statement

AbstractDiapause arrest in animals such as Caenorhabditis elegans is tightly regulated so that animals make appropriate developmental decisions amidst environmental challenges. Fully understanding diapause requires mechanistic insight of both entry and exit from the arrested state. While a steroid hormone pathway regulates the entry decision into Caenorhabditis elegans dauer diapause, its role in the exit decision is less clear. A complication to understanding steroid hormonal regulation of dauer has been the peculiar fact that steroid hormone mutants such as daf-9 form partial dauers under normal growth conditions. Here, we corroborate previous findings that daf-9 mutants remain capable of forming full dauers under unfavorable growth conditions, and we establish that the daf-9 partial dauer state is likely a partially exited dauer that has initiated but cannot complete the dauer exit decision. We show that the steroid hormone pathway is both necessary for and promotes complete dauer exit, and that the spatiotemporal dynamics of steroid hormone regulation during dauer exit resembles that of dauer entry. Overall, dauer entry and dauer exit are distinct developmental decisions that are both controlled by steroid hormone signaling.Summary StatementIn animals such as Caenorhabditis elegans, a steroid hormone pathway controls both the entry and exit decisions into and out of the developmentally arrested dauer state in response to environmental signaling.

scholarly journals Oocytes mount a noncanonical DNA damage response involving APC-Cdh1–mediated proteolysis

2020 ◽  
Vol 219 (4) ◽  
Author(s):  
Goutham Narayanan Subramanian ◽  
Jessica Greaney ◽  
Zhe Wei ◽  
Olivier Becherel ◽  
Martin Lavin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cyclin B1 ◽  
Chemical Induction ◽  
G2 Arrest ◽  
Damage Response ◽  
M Phase ◽  
Dependent Inhibition

In mitotic cells, DNA damage induces temporary G2 arrest via inhibitory Cdk1 phosphorylation. In contrast, fully grown G2-stage oocytes readily enter M phase immediately following chemical induction of DNA damage in vitro, indicating that the canonical immediate-response G2/M DNA damage response (DDR) may be deficient. Senataxin (Setx) is involved in RNA/DNA processing and maintaining genome integrity. Here we find that mouse oocytes deleted of Setx accumulate DNA damage when exposed to oxidative stress in vitro and during aging in vivo, after which, surprisingly, they undergo G2 arrest. Moreover, fully grown wild-type oocytes undergo G2 arrest after chemotherapy-induced in vitro damage if an overnight delay is imposed following damage induction. Unexpectedly, this slow-evolving DDR is not mediated by inhibitory Cdk1 phosphorylation but by APC-Cdh1–mediated proteolysis of the Cdk1 activator, cyclin B1, secondary to increased Cdc14B-dependent APC-Cdh1 activation and reduced Emi1-dependent inhibition. Thus, oocytes are unable to respond immediately to DNA damage, but instead mount a G2/M DDR that evolves slowly and involves a phosphorylation-independent proteolytic pathway.

scholarly journals FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2126
Author(s):  
Lorenzo Lafranchi ◽  
Erik Müllers ◽  
Dorothea Rutishauser ◽  
Arne Lindqvist
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cyclin B1 ◽  
Live Cells ◽  
Dependent Manner ◽  
Mitotic Entry ◽  
Protein Levels ◽  
Partial Inhibition ◽  
Damage Response ◽  
G2 Phase

Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populations within G2 phase. We employed mass spectroscopy to characterize changes in protein levels through an unperturbed G2 phase and validated that ATAD2 levels decrease in a proteasome-dependent manner. Comparing unperturbed cells with cells recovering from DNA damage, we note that at similar PLK1 activities, recovering cells contain higher levels of Cyclin B1 and increased phosphorylation of CDK1 targets. The increased Cyclin B1 levels are due to continuous Cyclin B1 production during a DNA damage response and are sustained until mitosis. Whereas partial inhibition of PLK1 suppresses mitotic entry more efficiently when cells recover from a checkpoint, partial inhibition of CDK1 suppresses mitotic entry more efficiently in unperturbed cells. Our findings provide a resource for proteome changes during G2 phase, show that the mitotic entry network is rewired during a DNA damage response, and suggest that the bottleneck for mitotic entry shifts from CDK1 to PLK1 after DNA damage.

Sign in / Sign up

Export Citation Format

Share Document