Cyclin A2/cyclin-dependent kinase 1–dependent phosphorylation of Top2a is required for S phase entry during retinal development in zebrafish

Terminal differentiation exerts a remarkably tight control on cell proliferation. However, the oncogenic products of DNA tumor viruses, such as adenovirus E1A, can force postmitotic cells to proliferate, thus representing a powerful tool to study progression into S phase. In this study, we identified the gene encoding Np95, a murine nuclear phosphoprotein, as an early target of E1A-induced transcriptional events. In terminally differentiated (TD) cells, the activation of Np95 was specifically induced by E1A, but not by overexpression of E2F-1 or of the cyclin E (cycE)–cyclin-dependent kinase 2 (cdk2) complex. In addition, the concomitant expression of Np95 and of cycE–cdk2 was alone sufficient to induce S phase in TD cells. In NIH-3T3 cells, the expression of Np95 was tightly regulated during the cell cycle, and its functional ablation resulted in abrogation of DNA synthesis. Thus, expression of Np95 is essential for S phase entry. Previous evidence suggested that E1A, in addition to its well characterized effects on the pRb/E2F-1 pathway, activates a parallel and complementary pathway that is also required for the reentry in S phase of TD cells (Tiainen, M., D. Spitkousky, P. Jansen-Dürr, A. Sacchi, and M. Crescenzi. 1996. Mol. Cell. Biol. 16:5302–5312). From our results, Np95 appears to possess all the characteristics to represent the first molecular determinant identified in this pathway.

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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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Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00249-10 ◽

2010 ◽

Vol 30 (21) ◽

pp. 5057-5070 ◽

Cited By ~ 17

Author(s):

David R. Croucher ◽

Danny Rickwood ◽

Carole M. Tactacan ◽

Elizabeth A. Musgrove ◽

Roger J. Daly

Keyword(s):

Cell Cycle ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Cell Cycle Progression ◽

Kinase Inhibitors ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Cycle Progression ◽

Fadu Cells ◽

Phase Entry

ABSTRACT The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

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Phosphorylation of the Cyclin-Dependent Kinase Inhibitor p21Cip1 on Serine 130 Is Essential for Viral Cyclin-Mediated Bypass of a p21Cip1-Imposed G1 Arrest

Molecular and Cellular Biology ◽

10.1128/mcb.26.6.2430-2440.2006 ◽

2006 ◽

Vol 26 (6) ◽

pp. 2430-2440 ◽

Cited By ~ 34

Author(s):

Annika Järviluoma ◽

Emma S. Child ◽

Grzegorz Sarek ◽

Papinya Sirimongkolkasem ◽

Gordon Peters ◽

...

Keyword(s):

Cell Cycle ◽

Kinase Inhibitor ◽

Primary Effusion Lymphoma ◽

S Phase ◽

G1 Arrest ◽

Cyclin Dependent Kinase ◽

Partial Restoration ◽

Viral Cyclin ◽

Phase Entry

ABSTRACT K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.

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Uncovering the role of APC-Cdh1 in generating the dynamics of S-phase onset

Molecular Biology of the Cell ◽

10.1091/mbc.e13-08-0480 ◽

2014 ◽

Vol 25 (4) ◽

pp. 441-456 ◽

Cited By ~ 24

Author(s):

Xi Yuan ◽

Jeyaraman Srividhya ◽

Thomas De Luca ◽

Ju-hyong E. Lee ◽

Joseph R. Pomerening

Keyword(s):

Kinase Inhibitor ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Cyclin E1 ◽

Stimulus Response ◽

Cyclin Dependent Kinase Inhibitor ◽

E1 Protein ◽

Reduced Frequency ◽

Phase Entry

Cdh1, a coactivator of the anaphase-promoting complex (APC), is a potential tumor suppressor. Cdh1 ablation promotes precocious S-phase entry, but it was unclear how this affects DNA replication dynamics while contributing to genomic instability and tumorigenesis. We find that Cdh1 depletion causes early S-phase onset in conjunction with increase in Rb/E2F1-mediated cyclin E1 expression, but reduced levels of cyclin E1 protein promote this transition. We hypothesize that this is due to a weakened cyclin-dependent kinase inhibitor (CKI)–cyclin-dependent kinase 2 positive-feedback loop, normally generated by APC-Cdh1–mediated proteolysis of Skp2. Indeed, Cdh1 depletion increases Skp2 abundance while diminishing levels of the CKI p27. This lowers the level of cyclin E1 needed for S-phase entry and delays cyclin E1 proteolysis during S-phase progression while corresponding to slowed replication fork movement and reduced frequency of termination events. In summary, using both experimental and computational approaches, we show that APC-Cdh1 establishes a stimulus–response relationship that promotes S phase by ensuring that proper levels of p27 accumulate during G1 phase, and defects in its activation accelerate the timing of S-phase onset while prolonging its progression.

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Glypican 1 Stimulates S Phase Entry and DNA Replication in Human Glioma Cells and Normal Astrocytes

Molecular and Cellular Biology ◽

10.1128/mcb.00238-13 ◽

2013 ◽

Vol 33 (22) ◽

pp. 4408-4421 ◽

Cited By ~ 12

Author(s):

Dianhua Qiao ◽

Kristy Meyer ◽

Andreas Friedl

Keyword(s):

Dna Replication ◽

Cyclin E ◽

Glioma Cells ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Human Glioma ◽

Human Glioma Cells ◽

Human Gliomas ◽

Phase Entry ◽

Dna Rereplication

Malignant gliomas are highly lethal neoplasms with limited treatment options. We previously found that the heparan sulfate proteoglycan glypican 1 (GPC1) is universally and highly expressed in human gliomas. In this study, we investigated the biological activity of GPC1 expression in both human glioma cells and normal astrocytesin vitro. Expression of GPC1 inactivates the G1/S checkpoint and strongly stimulates DNA replication. Constitutive expression of GPC1 causes DNA rereplication and DNA damage, suggesting a mutagenic activity for GPC1. GPC1 expression leads to a significant downregulation of the tumor suppressors pRb, Cip/Kip cyclin-dependent kinase inhibitors (CKIs), and CDH1, and upregulation of the pro-oncogenic proteins cyclin E, cyclin-dependent kinase 2 (CDK2), Skp2, and Cdt1. These GPC1-induced changes are accompanied by a significant reduction in all types of D cyclins, which is independent of serum supplementation. It is likely that GPC1 stimulates the so-called Skp2 autoinduction loop, independent of cyclin D-CDK4/6. Knockdown of Skp2, CDK2, or cyclin E, three key elements within the network modulated by GPC1, results in a reduction of the S phase and aneuploid fractions, implying a functional role for these regulators in GPC1-induced S phase entry and DNA rereplication. In addition, a significant activation of both the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways by GPC1 is seen in normal human astrocytes even in the presence of growth factor supplement. Both pathways are constitutively activated in human gliomas. The surprising magnitude and the mitogenic and mutagenic nature of the effect exerted by GPC1 on the cell cycle imply that GPC1 may play an important role in both glioma tumorigenesis and growth.

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A Skp2 autoinduction loop and restriction point control

The Journal of Cell Biology ◽

10.1083/jcb.200703034 ◽

2007 ◽

Vol 178 (5) ◽

pp. 741-747 ◽

Cited By ~ 36

Author(s):

Yuval Yung ◽

Janice L. Walker ◽

James M. Roberts ◽

Richard K. Assoian

Keyword(s):

S Phase ◽

Cyclin Dependent Kinase ◽

Embryonic Fibroblasts ◽

Papilloma Virus ◽

Restriction Point ◽

Pocket Proteins ◽

Serum Stimulation ◽

Point Control ◽

Phase Progression ◽

Phase Entry

We describe a self-amplifying feedback loop that autoinduces Skp2 during G1 phase progression. This loop, which contains Skp2 itself, p27kip1 (p27), cyclin E–cyclin dependent kinase 2, and the retinoblastoma protein, is closed through a newly identified, conserved E2F site in the Skp2 promoter. Interference with the loop, by knockin of a Skp2-resistant p27 mutant (p27T187A), delays passage through the restriction point but does not interfere with S phase entry under continuous serum stimulation. Skp2 knock down inhibits S phase entry in nontransformed mouse embryonic fibroblasts but not in human papilloma virus–E7 expressing fibroblasts. We propose that the essential role for Skp2-dependent degradation of p27 is in the formation of an autoinduction loop that selectively controls the transition to mitogen-independence, and that Skp2-dependent proteolysis may be dispensable when pocket proteins are constitutively inactivated.

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Faculty Opinions recommendation of Deubiquitinase USP37 is activated by CDK2 to antagonize APC(CDH1) and promote S phase entry.

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

10.3410/f.10823956.11737054 ◽

2011 ◽

Author(s):

Jean Wang

Keyword(s):

S Phase ◽

Phase Entry

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Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Cells ◽

10.3390/cells10030550 ◽

2021 ◽

Vol 10 (3) ◽

pp. 550

Author(s):

Indra A. Shaltiel ◽

Alba Llopis ◽

Melinda Aprelia ◽

Rob Klompmaker ◽

Apostolos Menegakis ◽

...

Keyword(s):

Dna Damage ◽

Normal Cell ◽

S Phase ◽

G1 Phase ◽

Anaphase Promoting Complex ◽

Inhibitory Effects ◽

Pocket Proteins ◽

Cyclin Dependent Kinases ◽

Independent Functions ◽

Phase Entry

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.

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

Science Advances ◽

10.1126/sciadv.abe3882 ◽

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.

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