Analysis of E1A domains involved in the enhancement of CDK2 activity

In somatic cells, cyclin E-cdk2 activity oscillates during the cell cycle and is required for the regulation of the G1/S transition. Cyclin E and its associated kinase activity remain constant throughout early sea urchin embryogenesis, consistent with reports from studies using several other embryonic systems. Here we have expanded these studies and show that cyclin E rapidly and selectively enters the sperm head after fertilization and remains concentrated in the male pronucleus until pronuclear fusion, at which time it disperses throughout the zygotic nucleus. We also show that cyclin E is not concentrated at the centrosomes but is associated with condensed chromosomes throughout mitosis for at least the first four cell cycles. Isolated mitotic spindles are enriched for cyclin E and cdk2, which are localized to the chromosomes. The chromosomal cyclin E is associated with active kinase during mitosis. We propose that cyclin E may play a role in the remodeling of the sperm head and re-licensing of the paternal genome after fertilization. Furthermore, cyclin E does not need to be degraded or dissociated from the chromosomes during mitosis; instead, it may be required on chromosomes during mitosis to immediately initiate the next round of DNA replication.

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c-Myc Overexpression Uncouples DNA Replication from Mitosis

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5339 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5339-5351 ◽

Cited By ~ 78

Author(s):

Qing Li ◽

Chi V. Dang

Keyword(s):

Mitotic Spindle ◽

Cyclin Dependent Kinase ◽

Wild Type ◽

Apoptotic Pathway ◽

Chromosomal Segregation ◽

Cdk2 Activity ◽

Murine Fibroblasts ◽

Immortalized Cells ◽

Primary Fibroblasts ◽

Massive Apoptosis

ABSTRACT c-myc has been shown to regulate G1/S transition, but a role for c-myc in other phases of the cell cycle has not been identified. Exposure of cells to colcemid activates the mitotic spindle checkpoint and arrests cells transiently in metaphase. After prolonged colcemid exposure, the cells withdraw from mitosis and enter a G1-like state. In contrast to cells in G1, colcemid-arrested cells have decreased G1 cyclin-dependent kinase activity and show hypophosphorylation of the retinoblastoma protein. We have found that overexpression of c-myc causes colcemid-treated human and rodent cells to become either apoptotic or polyploid by replicating DNA without chromosomal segregation. Although c-myc-induced polyploidy is not inhibited by wild-type p53 in immortalized murine fibroblasts, overexpression of c-myc in primary fibroblasts resulted in massive apoptosis of colcemid-treated cells. We surmise that additional genes are altered in immortalized cells to suppress the apoptotic pathway and allow c-myc-overexpressing cells to progress forward in the presence of colcemid. Our results also suggest that c-myc induces DNA rereplication in this G1-like state by activating CDK2 activity. These observations indicate that activation of c-myc may contribute to the genomic instability commonly found in human cancers.

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Identification of the functional domain of p21WAF1/CIP1 that protects cells from cisplatin cytotoxicity

AJP Renal Physiology ◽

10.1152/ajprenal.00101.2005 ◽

2005 ◽

Vol 289 (3) ◽

pp. F514-F520 ◽

Cited By ~ 39

Author(s):

Fang Yu ◽

Judit Megyesi ◽

Robert L. Safirstein ◽

Peter M. Price

Keyword(s):

Dominant Negative ◽

Nuclear Antigen ◽

Functional Domain ◽

Binding Domains ◽

Cdk2 Activity ◽

Localization Signal ◽

Induced Apoptosis ◽

Proliferating Cell

The p21 cyclin-dependent kinase (cdk) inhibitor protects cells from cisplatin cytotoxicity in vivo and in vitro. However, the mechanism of protection is not known. Separate p21 domains are known to interact with several different proteins having proapoptotic functions. To investigate the mechanism of protection by p21, we have constructed adenoviruses encoding the different domains of p21. We were able to localize the protective activity to a region of 54 amino acids containing the cyclin-cdk interacting moiety. Other protein binding domains of p21, including the NH2-terminal procaspase-3 interactive region and the COOH-terminal region containing the proliferating cell nuclear antigen binding domain and the nuclear localization signal, had little protective effect on cisplatin cytotoxicity. The dependence of cisplatin cytotoxicity on cdk2 activity was also demonstrated because 1) cisplatin caused a marked increase in cdk2 activity, which was prevented by the p21 expression adenovirus, and 2) a cdk2 dominant-negative adenovirus also protected cells from cisplatin-induced apoptosis. Thus the data suggest that the mechanism of p21 protection is by direct inhibition of cdk2 activity and that cisplatin-induced apoptosis is caused by a cdk2-dependent pathway.

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Intra-S-Phase Checkpoint Activation by Direct CDK2 Inhibition

Molecular and Cellular Biology ◽

10.1128/mcb.24.14.6268-6277.2004 ◽

2004 ◽

Vol 24 (14) ◽

pp. 6268-6277 ◽

Cited By ~ 41

Author(s):

Yonghong Zhu ◽

Carmen Alvarez ◽

Ronald Doll ◽

Hirokazu Kurata ◽

Xiao Min Schebye ◽

...

Keyword(s):

Genomic Stability ◽

S Phase ◽

Cell Cycle Checkpoints ◽

Minichromosome Maintenance ◽

Checkpoint Activation ◽

Cdk2 Activity ◽

A Cell ◽

Dependent Pathway ◽

Number Of Cells ◽

S Phase Checkpoint

ABSTRACT To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.

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Involvement of p27kip1 and cyclin D3 in the regulation of cdk2 activity during skeletal muscle differentiation

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/s0167-4889(00)00064-1 ◽

2000 ◽

Vol 1497 (2) ◽

pp. 175-185 ◽

Cited By ~ 10

Author(s):

Cindy Y Chu ◽

Robert W Lim

Keyword(s):

Skeletal Muscle ◽

Muscle Differentiation ◽

Cyclin D3 ◽

Skeletal Muscle Differentiation ◽

Cdk2 Activity

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TBP-associated factor 1 overexpression induces tolerance to Doxorubicin in confluent H9c2 cells by an increase in cdk2 activity and cyclin E expresion

Molecular and Cellular Biochemistry ◽

10.1023/b:mcbi.0000021347.65073.10 ◽

2004 ◽

Vol 259 (1/2) ◽

pp. 71-81 ◽

Cited By ~ 1

Author(s):

Nicolas Servant ◽

Daniela Marcantonio ◽

John P.H. Th'ng ◽

Lorraine E. Chalifour

Keyword(s):

Cyclin E ◽

H9c2 Cells ◽

Associated Factor ◽

Cdk2 Activity

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Cell-cycle arrest in Jurkat leukaemic cells: a possible role for docosahexaenoic acid

Biochemical Journal ◽

10.1042/bj20021667 ◽

2003 ◽

Vol 371 (2) ◽

pp. 621-629 ◽

Cited By ~ 51

Author(s):

Rafat A. SIDDIQUI ◽

Laura J. JENSKI ◽

Kevin A. HARVEY ◽

Jacqueline D. WIESEHAN ◽

William STILLWELL ◽

...

Keyword(s):

Cell Cycle ◽

Docosahexaenoic Acid ◽

Cell Cycle Arrest ◽

Protein Phosphatase ◽

Cyclin A ◽

Low Doses ◽

Induce Cell Cycle Arrest ◽

Cycle Arrest ◽

Cdk2 Activity ◽

Leukaemic Cells

Docosahexaenoic acid (DHA) is known to have anti-cancer activities by mechanisms that are not well understood. In the present study, we test one possible pathway for DHA action in Jurkat leukaemic cells. Low doses of DHA (10μM) are shown to induce cell-cycle arrest, whereas higher doses are cytotoxic. However, when cells that were pre-treated with 10μM DHA are given an additional 10μM DHA dose, cell viability rapidly decreases. Immunoblotting reveals that repeated low doses of DHA results in activation of caspase 3, implying induction of apoptosis. DHA (10μM) is shown to increase ceramide levels after 6h of incubation and, after 24h, the cells appear to be arrested in S phase. With DHA, the amount of phosphorylated retinoblastoma protein (pRb) decreases significantly. Western blot analysis also shows that DHA greatly reduces the level of cyclin A, while increasing the level of p21 WAF1, a cellular inhibitor of cyclin A/cyclin-dependent kinase 2 (cdk2) activity. Furthermore, the observed DHA-induced doubling of the ratio of hypophosphorylated pRb (hypo-pRb) to total pRb is inhibited by tautomycin and phosphatidic acid (PA), known inhibitors of protein phosphatase 1 (PP1), and by the PP2 inhibitor okadaic acid. The present study demonstrates one possible connected pathway for DHA action. By this pathway, low doses of DHA increase ceramide levels, which leads to inhibition of cdk2 activity and stimulation of PP1 and PP2A. The net effect of cdk2 inhibition and protein phosphatase activation is an inhibition of pRb phosphorylation, consequently arresting Jurkat cell growth.

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