scholarly journals p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.

1997 ◽  
Vol 8 (9) ◽  
pp. 1815-1827 ◽  
Author(s):  
P Shiyanov ◽  
S Hayes ◽  
N Chen ◽  
D G Pestov ◽  
L F Lau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Culture Medium ◽  
Cyclin E ◽  
Cyclin A ◽  
Cyclin Dependent Kinase ◽  
Binding Motifs ◽  
Cyclin Dependent Kinases ◽  
Cell Cycle Inhibition ◽  
A Cell

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

scholarly journals Differential Control of Transcription by DNA-bound Cyclins

2001 ◽  
Vol 12 (7) ◽  
pp. 2207-2217 ◽  
Author(s):  
Tae-You Kim ◽  
William G. Kaelin
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cyclin E ◽  
Cyclin A ◽  
Dependent Manner ◽  
Dna Binding Domain ◽  
Transcriptional Modulators ◽  
A Cell ◽  
Cycle Dependent ◽  
Differential Control

Different cyclins mediate different cell-cycle transitions. Some cyclins, such as cyclin A and cyclin E, form stable complexes with proteins that bind directly or indirectly to DNA and thus might be recruited to certain regions of the genome at specific times in the cell cycle. Furthermore, cyclins contain structural motifs that are also present in known transcriptional modulators. We found that cyclin A is a potent transcriptional repressor and cyclin E is a potent transcriptional activator when bound to DNA via a heterologous DNA binding domain. The former activity was linked to the integrity of the cyclin A cyclin fold, whereas the latter activity related to the ability of cyclin E to activate cdk2 and recognize substrates. Furthermore, we found that cyclin E, but not cyclin A, activated transcription in a cell-cycle–dependent manner when present in physiological concentrations as an unfused protein. These results suggest that cyclin A and cyclin E intrinsically differ with respect to their ability to modulate transcription when tethered to DNA.

scholarly journals NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells

1999 ◽  
Vol 276 (5) ◽  
pp. H1450-H1459 ◽  
Author(s):  
Ram V. Sharma ◽  
Enqing Tan ◽  
Shengyun Fang ◽  
Milind V. Gurjar ◽  
Ramesh C. Bhalla
Keyword(s):  
Nitric Oxide ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Cyclin E ◽  
Cyclin A ◽  
Nuclear Antigen ◽  
Enos Gene ◽  
Cyclin Dependent Kinase

The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene ( eNOS) into VSM cells significantly inhibited ( P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited ( P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOStransfer. eNOS transfer did not decrease PDGF-β receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation.

scholarly journals The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

1996 ◽  
Vol 16 (4) ◽  
pp. 1335-1341 ◽  
Author(s):  
W Poluha ◽  
D K Poluha ◽  
B Chang ◽  
N E Crosbie ◽  
C M Schonhoff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Neuronal Differentiation ◽  
Kinase Inhibitor ◽  
Cyclin E ◽  
Neuroblastoma Cells ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinases ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Differentiated Cells

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.

Inhibitors of Cyclin-Dependent Kinase 1/2 for Anticancer Treatment

2020 ◽  
Vol 16 (3) ◽  
pp. 307-325
Author(s):  
Jiajia Mou ◽  
Danghui Chen ◽  
Yanru Deng
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Cyclin Dependent Kinase ◽  
Allosteric Inhibitors ◽  
Cyclin Dependent Kinases ◽  
Chemical Structures ◽  
Structure Activity ◽  
Endogenous Inhibitors ◽  
Anticancer Treatment ◽  
Anticancer Drug Development

Background: The cell cycle is regulated by cyclin-dependent kinases (CDKs) and their cognate cyclins, along with their endogenous inhibitors (CDKIs). CDKs act as central regulators in this process. Different CDKs play relevant roles in different phases. Among all CDKs, CDK1 is indispensible, which can drive all events that are required in the cell cycle in the absence of interphase CDKs (CDK2, CDK3, CDK4 and CDK6). So, CDK1 is an attractive target for anticancer drug development. Methods: CDK1 and CDK2 have 89.19% similar residues and 74.32% identical residues, their structures especially the ATP-binding sites are of great similarity. So, it is difficult to inhibit CDK1 and CDK2 individually. In this review, recent advances about CDK1/2 inhibitors were summarized. The chemical structures of different classes of CDK1/2 inhibitors and their structure activity are presented. Results: 19 kinds of CDK1/2 or CDK1 inhibitors with different scaffolds, including CDK2 allosteric inhibitors, were summarized. Some inhibitors are nature derived, for example, phenanthrene derivatives, nortopsentin derivatives, variolin B derivatives and meridians. Conclusion: Nature products, especially marine ones are potential resources for CDK1 inhibitors development. The findings of CDK2 allosteric inhibitors open an avenue to the discovery of novel selective CDK1 or other CDKs allosteric inhibitors.

scholarly journals Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

1999 ◽  
Vol 19 (1) ◽  
pp. 646-656 ◽  
Author(s):  
Christian Voitenleitner ◽  
Christoph Rehfuess ◽  
Melissa Hilmes ◽  
Lynda O’Rear ◽  
Pao-Chi Liao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Polymerase ◽  
Cyclin E ◽  
Cyclin A ◽  
Human Cells ◽  
Dependent Manner ◽  
Dna Polymerase Α ◽  
A Cell ◽  
Cycle Dependent

ABSTRACT DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

scholarly journals p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.

1997 ◽  
Vol 17 (7) ◽  
pp. 3566-3579 ◽  
Author(s):  
M S Woo ◽  
I Sánchez ◽  
B D Dynlacht
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Kinase Activity ◽  
Cyclin E ◽  
Critical Role ◽  
Cyclin A ◽  
Terminal Region ◽  
Cyclin Dependent Kinase ◽  
Amino Terminal

The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases.

Cyclin A, cell cycle control and oncogenesis

1991 ◽  
Vol 3 (4) ◽  
pp. 267-277 ◽  
Author(s):  
Michele Pagano ◽  
Giulio Draetta
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Control ◽  
Cyclin A ◽  
A Cell

scholarly journals Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases

2001 ◽  
Vol 21 (15) ◽  
pp. 4868-4874 ◽  
Author(s):  
James A. Wohlschlegel ◽  
Brian T. Dwyer ◽  
David Y. Takeda ◽  
Anindya Dutta
Keyword(s):  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Cyclin E ◽  
Cyclin A ◽  
Binding Motif ◽  
Binding Studies ◽  
Alanine Scanning Mutagenesis ◽  
Cyclin Dependent Kinases

ABSTRACT Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.

scholarly journals Cyclin E overexpression in relapsed adult acute lymphoblastic leukemias of B-cell lineage

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3360-3367 ◽  
Author(s):  
R Scuderi ◽  
KA Palucka ◽  
K Pokrovskaja ◽  
M Bjorkholm ◽  
KG Wiman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Lymphoblastic Leukemia ◽  
Cyclin E ◽  
Cell Lineage ◽  
Cyclin A ◽  
Mean Value ◽  
Peripheral Blood Mononuclear ◽  
Protein Levels

Using Western blot analysis, we examined cyclin E and cyclin A protein levels in 19 patients with acute lymphoblastic leukemia ([ALL] 15 B-ALL and four T-ALL). Whereas normal, nonproliferating peripheral blood mononuclear cells (PBMCs) expressed low levels of the 50-kD cyclin E, ALL blasts in the peripheral blood, although showing low-level or no proliferation as judged by FACS/cell-cycle analysis and cyclin A protein levels, expressed high levels of cyclin E, with a mean value similar to that of the proliferating Burkitt's lymphoma cell line, Akata. The accumulation of a protein shown to shorten the G1 phase of the cell cycle, cyclin E, in growth-delayed leukemic blasts may reflect the malignant status of these cells. Before treatment, B-ALL cells expressed predominantly the 50-kD cyclin E. T-ALL samples displayed the 50-kD cyclin E protein and a smaller, approximately 43-kD cyclin E species. In paired B-ALL samples taken before treatment and at relapse, we found a significant overexpression of the 50-kD protein in relapsed samples (P < .006), plus the presence of up to four additional smaller- molecular-weight species of cyclin E, illustrating clear diagnosis versus relapse differences. Cyclin E expression in ALL blasts may correlate to the relative malignant status of the cells, with higher protein levels reflecting a more advanced stage of the disease and a greater potential to proliferate under permissive conditions.

scholarly journals Structure-function relationships of the yeast cyclin-dependent kinase Pho85.

1995 ◽  
Vol 15 (10) ◽  
pp. 5482-5491 ◽  
Author(s):  
R C Santos ◽  
N C Waters ◽  
C L Creasy ◽  
L W Bergman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structure Function ◽  
Cell Cycle Progression ◽  
Substrate Recognition ◽  
The Other ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Induced Mutagenesis

The PHO85 gene of Saccharomyces cerevisiae encodes a cyclin-dependent kinase involved in both transcriptional regulation and cell cycle progression. Although a great deal is known concerning the structure, function, and regulation of the highly homologous Cdc28 protein kinase, little is known concerning these relationships in regard to Pho85. In this study, we constructed a series of Pho85-Cdc28 chimeras to map the region(s) of the Pho85 molecule that is critical for function of Pho85 in repression of acid phosphatase (PHO5) expression. Using a combination of site-directed and ethyl methanesulfonate-induced mutagenesis, we have identified numerous residues critical for either activation of the Pho85 kinase, interaction of Pho85 with the cyclin-like molecule Pho80, or substrate recognition. Finally, analysis of mutations analogous to those previously identified in either Cdc28 or cdc2 of Schizosaccharomyces pombe suggested that the inhibition of Pho85-Pho80 activity in mechanistically different from that seen in the other cyclin-dependent kinases.

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