scholarly journals Periodic Expression of the Cyclin-dependent Kinase Inhibitor p57Kip2 in Trophoblast Giant Cells Defines a G2-like Gap Phase of the Endocycle

2000 ◽  
Vol 11 (3) ◽  
pp. 1037-1045 ◽  
Author(s):  
Naka Hattori ◽  
Tyler C. Davies ◽  
Lynn Anson-Cartwright ◽  
James C. Cross
Keyword(s):  
Dna Replication ◽  
Protein Targeting ◽  
Kinase Inhibitor ◽  
Phosphorylation Site ◽  
Giant Cells ◽  
S Phase ◽  
Stable Form ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Trophoblast Giant Cells

Endoreduplication is an unusual form of cell cycle in which rounds of DNA synthesis repeat in the absence of intervening mitoses. How G1/S cyclin-dependent kinase (Cdk) activity is regulated during the mammalian endocycle is poorly understood. We show here that expression of the G1/S Cdk inhibitor p57Kip2 is induced coincidentally with the transition to the endocycle in trophoblast giant cells.Kip2 mRNA is constitutively expressed during subsequent endocycles, but the protein level fluctuates. In trophoblast giant cells synchronized for the first few endocycles, the p57Kip2 protein accumulates only at the end of S-phase and then rapidly disappears a few hours before the onset of the next S-phase. The protein becomes stabilized by mutation of a C-terminal Cdk phosphorylation site. As a consequence, introduction of this stable form of p57Kip2 into giant cells blocks S-phase entry. These data imply that p57Kip2 is subject to phosphorylation-dependent turnover. Surprisingly, although this occurs in endoreduplicating giant cells, p57Kip2 is stable when ectopically expressed in proliferating trophoblast cells, indicating that these cells lack the mechanism for protein targeting and/or degradation. These data show that the appearance of p57Kip2punctuates the completion of DNA replication, whereas its turnover is subsequently required to initiate the next round of endoreduplication in trophoblast giant cells. Cyclical expression of a Cdk inhibitor, by terminating G1/S Cdk activity, may help promote the resetting of DNA replication machinery.

scholarly journals Cell cycle inertia underlies a bifurcation in cell fates after DNA damage

2021 ◽  
Vol 7 (3) ◽  
pp. eabe3882
Author(s):  
Jenny F. Nathans ◽  
James A. Cornwell ◽  
Marwa M. Afifi ◽  
Debasish Paul ◽  
Steven D. Cappell
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cell Tracking ◽  
Time Lapse ◽  
S Phase ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Cell Fates ◽  
Damage Response ◽  
Time Lapse Imaging

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

scholarly journals Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sabrina F Mansilla ◽  
Agustina P Bertolin ◽  
Valérie Bergoglio ◽  
Marie-Jeanne Pillaire ◽  
Marina A González Besteiro ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Genomic Stability ◽  
S Phase ◽  
Fragile Sites ◽  
Dna Lesions ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Independent Manner ◽  
Genomic Regions

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

scholarly journals SGK1-Sensitive Regulation of Cyclin-Dependent Kinase Inhibitor 1B (p27) in Cardiomyocyte Hypertrophy

2015 ◽  
Vol 37 (2) ◽  
pp. 603-614 ◽  
Author(s):  
Jakob Voelkl ◽  
Tatsiana Castor ◽  
Katharina Musculus ◽  
Robert Viereck ◽  
Sobuj Mia ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Kinase Inhibitor ◽  
Protein Localization ◽  
Phosphorylation Site ◽  
Cardiomyocyte Hypertrophy ◽  
Mrna Levels ◽  
Cyclin Dependent Kinase ◽  
Transcript Levels ◽  
Hypertrophic Response ◽  
Cyclin Dependent Kinase Inhibitor

Background/Aims: The serum- and glucocorticoid-inducible kinase SGK1 participates in the orchestration of cardiac hypertrophy and remodeling. Signaling linking SGK1 activity to cardiac remodeling is, however, incompletely understood. SGK1 phosphorylation targets include cyclin-dependent kinase inhibitor 1B (p27), a protein which suppresses cardiac hypertrophy. The present study explored how effects of SGK1 on nuclear p27 localization might modulate the hypertrophic response in cardiomyocytes. Methods: Experiments were performed in HL-1 cardiomyocytes and in SGK1-deficient (sgk1-/-) and corresponding wild-type (sgk1+/+) mice following pressure overload by transverse aortic constriction (TAC). Transcript levels were quantified by RT-PCR, protein abundance by Western blotting and protein localization by confocal microscopy. Results: In HL-1 cardiomyocytes, overexpression of constitutively active SGK1 (SGK1S422D) but not of inactive SGK1 (SGK1K127N) increased significantly the cell size and transcript levels encoding Acta1, a molecular marker of hypertrophy. Those effects were paralleled by almost complete relocation of p27 in the cytoplasm. Treatment of HL-1 cardiomyocytes with isoproterenol was followed by up-regulation of SGK1 expression. Moreover, isoproterenol treatment stimulated the hypertrophic response and was followed by disappearance of p27 from the nuclei, effects prevented by the SGK1 inhibitor EMD638683. The effect of SGK1S422D overexpression on Acta1 mRNA levels was disrupted by overexpression of p27 and of the p27T197A mutant lacking the SGK1 phosphorylation site, but not of the phosphomimetic p27T197D mutant. In sgk1+/+ mice, TAC increased significantly SGK1 and Acta1 mRNA levels and decreased the nuclear to cytoplasmic protein ratio of p27 in cardiac tissue, effects blunted in the sgk1-/- mice. Conclusion: SGK1-induced hypertrophy of cardiomyocytes involves p27 phosphorylation at T197, which fosters cytoplasmic p27 localization.

scholarly journals Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

scholarly journals Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

2015 ◽  
Vol 290 (35) ◽  
pp. 21536-21552 ◽  
Author(s):  
Debduti Datta ◽  
Kumaraswamy Anbarasu ◽  
Suryaraja Rajabather ◽  
Rangasamy Sneha Priya ◽  
Pavitra Desai ◽  
...  
Keyword(s):  
Phase Transition ◽  
Kinase Inhibitor ◽  
Binding Protein ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Gtp Binding Protein ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Gtp Binding

scholarly journals Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

2010 ◽  
Vol 21 (19) ◽  
pp. 3421-3432 ◽  
Author(s):  
Donna Garvey Brickner ◽  
Jason H. Brickner
Keyword(s):  
Cell Cycle ◽  
Nuclear Periphery ◽  
S Phase ◽  
Nuclear Pore ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Initiation Of Dna Replication ◽  
Cdk Phosphorylation ◽  
Inducible Genes ◽  
Active Genes

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.

scholarly journals Coupled Transcriptional and Translational Control of Cyclin-Dependent Kinase Inhibitor p18INK4cExpression during Myogenesis

1998 ◽  
Vol 18 (4) ◽  
pp. 2334-2343 ◽  
Author(s):  
Dawn E. Phelps ◽  
Kuang-Ming Hsiao ◽  
Yan Li ◽  
Nanpin Hu ◽  
David S. Franklin ◽  
...  
Keyword(s):  
Translational Control ◽  
Kinase Inhibitor ◽  
Myogenic Differentiation ◽  
Protein A ◽  
Terminal Differentiation ◽  
C2c12 Cells ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
C2c12 Myoblasts ◽  
Exon 1

ABSTRACT Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18 INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18 INK4c gene expresses two mRNA transcripts—a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. As C2C12 cells are induced to differentiate into permanently arrested myotubes, the abundance of the p18(L) transcript decreases. The smaller p18(S) transcript expressed from a downstream promoter becomes detectable by 12 h postinduction and is the predominant transcript expressed in terminally differentiated myotubes. Both transcripts contain coding exons 2 and 3, but p18(L) uniquely contains an additional noncoding 1.2-kb exon, exon 1, corresponding exclusively to the 5′ untranslated region (5′ UTR). The expression pattern of the shorter p18(S) transcript, but not that of the longer p18(L) transcript, correlates with terminal differentiation of muscle, lung, liver, thymus, and eye lens cells during mouse embryo development. The presence of the long 5′ UTR in exon 1 attenuated the translation of p18(L) transcript, while its absence from the shorter p18(S) transcript resulted in significantly more efficient translation of the p18 protein. Our results demonstrate that during terminal muscle cell differentiation, induction of the p18 protein is regulated by promoter switching coupled with translational control.

scholarly journals Interplay between S-Cyclin-dependent Kinase and Dbf4-dependent Kinase in Controlling DNA Replication through Phosphorylation of Yeast Mcm4 N-Terminal Domain

2008 ◽  
Vol 19 (5) ◽  
pp. 2267-2277 ◽  
Author(s):  
Alain Devault ◽  
Elisabeth Gueydon ◽  
Etienne Schwob
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Loss Of Function ◽  
Negative Effects ◽  
Origin Firing ◽  
Synthetic Lethal ◽  
Terminal Domain ◽  
Firing Efficiency

Cyclin-dependent (CDK) and Dbf4-dependent (DDK) kinases trigger DNA replication in all eukaryotes, but how these kinases cooperate to regulate DNA synthesis is largely unknown. Here, we show that budding yeast Mcm4 is phosphorylated in vivo during S phase in a manner dependent on the presence of five CDK phosphoacceptor residues within the N-terminal domain of Mcm4. Mutation to alanine of these five sites (mcm4-5A) abolishes phosphorylation and decreases replication origin firing efficiency at 22°C. Surprisingly, the loss of function mcm4-5A mutation confers cold and hydroxyurea sensitivity to DDK gain of function conditions (mcm5/bob1 mutation or DDK overexpression), implying that phosphorylation of Mcm4 by CDK somehow counteracts negative effects produced by ectopic DDK activation. Deletion of the S phase cyclins Clb5,6 is synthetic lethal with mcm4-5A and mimics its effects on DDK up mutants. Furthermore, we find that Clb5 expressed late in the cell cycle can still suppress the lethality of clb5,6Δ bob1 cells, whereas mitotic cyclins Clb2, 3, or 4 expressed early cannot. We propose that the N-terminal extension of eukaryotic Mcm4 integrates regulatory inputs from S-CDK and DDK, which may play an important role for the proper assembly or stabilization of replisome–progression complexes.

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