Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multiple species of G1 cyclins and cyclin-dependent kinases are induced sequentially during G1 phase, and the expression of cyclin A and cdc2 genes is subsequently induced at the G1/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5'-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, which contains the E box, SpI, and E2F motifs. The expression of 5' sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses revealed that the enhancer sequence, AAGTTACAAATA, located from -276 to -265, confers strong inducibility on the basal promoter at the G1/S boundary. The base substitutions introduced into the motifs of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G1/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G1 phase.
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PMID:The G1/S boundary-specific enhancer of the rat cdc2 promoter. 773 68

Expression of c-myc with constitutively active mutants of the ras gene results in the cooperative transformation of primary fibroblasts, although the precise mechanism by which these genes cooperate is unknown. Since c-Myc has been shown to function as a transcriptional activator, we have examined the ability of c-Myc and activated Ras (H-RasV-12) to cooperatively induce the promoter activity of cdc2, a gene which is critical for cell cycle progression. Microinjection of expression constructs encoding H-RasV-12 and c-Myc along with a cdc2 promoter-luciferase reporter plasmid into quiescent cells led to an increase in cdc2 promoter activity approximately 30 h after injection, a period which coincides with the S-to-G2/M transition in these cells. Expression of H-RasV-12 alone weakly activated the cdc2 promoter, while expression of c-Myc alone had no effect. Mutants of c-Myc lacking either the leucine zipper dimerization domain or the phosphoacceptor site Ser-62 could not cooperate with H-RasV-12 to induce the cdc2 promoter. These mutants also lacked the ability to cooperate with H-RasV-12 to stimulate DNA synthesis. Deletion analysis identified a distinct region of the cdc2 promoter which was required for c-Myc responsiveness. Taken together, these observations suggest a mechanistic link between the molecular activities of c-Myc and Ras and induction of the cell cycle regulator Cdc2.
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PMID:c-Myc cooperates with activated Ras to induce the cdc2 promoter. 806 6

The CDK-inhibitor p21WAF1/CIP1 has been implicated as a growth arrest mediator in p53-tumour suppression, cellular senescence and terminal differentiation. Cell type specific differences in p53-independent p21 expression and cell cycle arrest were found following treatment of human tumour cell lines with serum, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or okadaic acid (OA). TPA induced p21 in ML1, K562 and HL60 leukemia cells, whereas OA induced p21 in SW480 and GM4723 carcinoma cells as well as in leukemic cells. In addition, TPA- and serum- but not OA-induced cell cycle arrest was reversed upon return of p21 to basal levels. To further investigate the mechanisms underlying p53-independent regulation of p21, the transcription inhibitor, Actinomycin D (AMD), was used to block p21 expression. The results showed a complete inhibition of p21 mRNA and protein induction by TPA or adriamycin but little effect on p21 mRNA induced by OA in the presence of AMD. These results suggested that TPA-induced p21 expression requires transcription initiation, while a post-transcriptional mechanism may be involved in OA-induction as well. Transient transfection assays with p21 promoter-luciferase reporters and TPA or OA treatment further confirmed that TPA, and to a lesser extent, OA, initiated transcription of p21. Finally, the protein kinase C inhibitor, staurosporine, was found to interfere with p21 induction and prevent cell cycle arrest following treatment with TPA but not OA, suggesting a requirement for PKC in TPA activation of p21 expression.
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PMID:Regulation of p21WAF1/CIP1 expression by p53-independent pathways. 862 72

Histone gene expression is restricted to the S phase of the cell cycle. Control is mediated by a complex network of sequence-specific DNA-binding factors and protein-protein interactions in response to cell cycle progression. To further investigate the regulatory functions that are associated at the transcriptional level, we analyzed the regulation of a replication-dependent human H2A.1-H2B.2 gene pair. We found that transcription factor E2F binds specifically to an E2F recognition motif in the H2A.1 promoter region. Activation of the H2A.1 promoter by E2F-1 was shown by use of luciferase reporter constructs of the intergenic promoter region. Overexpression of the human retinoblastoma suppressor gene product RB suppressed E2F-1 mediated transcriptional activation, indicating an E2F-dependent regulation of promoter activity during the G1-to-S-phase transition. Furthermore, the activity of the H2A.1 promoter was also downregulated by overexpression of the RB-related p107, a protein that has been detected in S-phase-specific protein complexes of cyclin A, E2F, and cdk2. In synchronized HeLa cells, expression of luciferase activity was induced at the beginning of DNA synthesis and was dependent on the presence of an E2F-binding site in the H2A.1 promoter. Together with the finding that E2F-binding motifs are highly conserved in H2A promoters of other species, our results suggest that E2F plays an important role in the coordinate regulation of S-phase-specific histone gene expression.
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PMID:The E2F transcription factor activates a replication-dependent human H2A gene in early S phase of the cell cycle. 862 55

MCF-7 human breast cancer cells express functional estrogen receptor and grow in response to estrogen stimulation. G(1)-synchronized MCF-7 cells, made quiescent by exposure to the HMG-CoA reductase inhibitor Simvastatin in estrogen-free medium, readily resume cell cycle progression upon stimulation with 17beta-estradiol (E(2)), even under conditions where polypeptide growth factor-triggered signal transduction pathways are inhibited by the continuous presence of Simvastatin in the culture medium. Under these conditions, cyclin D(1) gene transcription is transiently induced within the first 1-9 h of stimulation, as shown by the accumulation of cyclin D(1) mRNA and protein (p36(D(1))) in the cell and by enhanced expression of stably transfected D(1) promoter-luciferase hybrid genes. Estrogen-induced p36(D(1)) associates readily with p32(cdk2) and p34(cdk4), but not with p31(cdk5), which is however abundantly expressed in these cells. Only p36(D(1))-p34(cdk4) complexes are activated by E(2), as detected in cell extracts by immunoprecipitation with anti-D(1) antibodies followed by assessment of phosphotransferase activity toward the retinoblastoma (Rb) gene product and by analysis of p105(Rb) phosphorylation in vivo. An estrogen-responsive regulatory region has been mapped within the first 944 bp upstream of the transcriptional startsite of the human D(1) gene. Sequence analysis of this DNA region reveals that the cis-acting elements responsive to estrogen are likely to be different in this case from the canonical EREs.
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PMID:17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells. 864 71

Cyclin E controls progression through the G1 phase of the cell cycle in mammalian fibroblasts and potentially in many other cell types. Cyclin E is a rate-limiting activator of cdk2 kinase in late G1. The abundance of cyclin E is controlled by phase-specific fluctuations in the mRNA level; in mammalian fibroblasts, mRNA is not detected under conditions of serum starvation and is accumulated upon serum stimulation, with expression starting in mid-G1. Here, we report the cloning of the murine cyclin E promoter. We isolated a 3.8-kb genomic fragment that contains several transcriptional start sites and confers cell cycle regulation on a luciferase reporter gene. This fragment also supports transcriptional activation by adenovirus E1A, a known upstream regulator of cyclin E gene expression. An E2F binding site which is required for G1-specific activation of the cyclin E promoter in synchronized NIH 3T3 cells was identified in this fragment.
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PMID:Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter. 866 55

Loss of adhesion of NRK fibroblasts to an appropriate surface leads to cell cycle arrest in late G1 and failure to produce cyclin A. Previously, we showed that adhesion-dependent expression of cyclin A is transcriptionally regulated. In an effort to identify elements of the adhesion-mediated signal transduction cascade upstream of cyclin A activation, we investigated the expression of cyclin E and its associated kinase activity in adherent and suspended NRK cells. Expression of cyclin E was found to be unaffected by suspension. However, cyclin E complexes immunoprecipitated from extracts prepared from NRK cells 12 h after release from G0 arrest were found to be catalytically inactive in suspended but not in adherent cells. This suspension-induced inhibition of cyclin E-associated kinase activity was not observed in NRK cells transformed by a c-Ha-ras oncogene containing a G12V mutation. When G0-synchronized NRK cells were transfected with a cyclin A promoter:luciferase reporter construct along with expression vectors for either wild-type cdk2 or a dominant-negative cdk2 mutant, transcriptional activation of cyclin A was found to be dependent on catalytically active cdk2. Inhibition of cyclin E/cdk2 complexes has frequently been attributed to association of the cdk inhibitors p21(Cip1) and p27(Kip1). However, no differences between adherent and suspended cells could be observed for either expression or cdk2 association of p21(Cip1) or p27(Kip1), nor were any proteins specifically associated with cdk2 or cyclin E in immunoprecipitates from metabolically labeled cell extracts. These results define a pathway through which an adhesion-generated signal controls cyclin A expression by modulating cyclin E/cdk2 activity.
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PMID:Adhesion-dependent control of cyclin E/cdk2 activity and cell cycle progression in normal cells but not in Ha-ras transformed NRK cells. 894 Feb 52

UCN-01 (7-hydroxyl-staurosporine) was originally isolated as a Ca2+- and phospholipid-dependent protein kinase C selective inhibitor and now is being developed as an anticancer agent. Results from our and other laboratories have suggested that UCN-01 induces preferential G1-phase accumulation in several human tumor cell lines tested. To elucidate this mechanism, we examined the effects of UCN-01 on several cell cycle-regulatory proteins critical for G1-S-phase transition in p53-mutated human epidermoid carcinoma A431 cells. After 24 h exposure at around 50% growth-inhibitory concentrations (IC50s), 260 and 520 nM, UCN-01 induced the accumulation of pRb (the dephosphorylated retinoblastoma protein form). The protein expression of cyclin A but not cyclin E was markedly reduced and that of cyclin D1 was partially reduced under the same condition. UCN-01 also showed the concentration-dependent inhibitions of the activity of cyclin-dependent kinase 2 (CDK2) using histone H1 and pRb as substrates in vitro (IC50, 530 and 640 nM, respectively). In addition, CDK2 activities of the cells pretreated with UCN-01 for 24 h at 260 and 520 nM were markedly inhibited, giving IC50s of far less than 260 nM. When the same cell lysates were analyzed by Western blotting for CDK2, the lower band (e.g., active and phosphorylated CDK2) was remarkably reduced, in accordance with the reduced activity. Furthermore, UCN-01 induced the expression of the CDK inhibitor p21 protein and its complex formation with CDK2 after 24 h exposure at 260 and 520 nM, whereas the expression level was very low or undetectable in untreated or DNA-damaged cells. The increase of p21 mRNA levels was also induced under the same condition. UCN-01 further increased luciferase activities in A431 cells transiently transfected with p21 promoter-luciferase reporter plasmid after 24 h exposure at 260 and 520 nM. UCN-01 also increased the expression of the CDK inhibitor p27 protein after 24 h exposure at 260 and 520 nM. These results suggest that G1-phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitors p21 and p27.
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PMID:G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells. 910 51

To study the effect of several cyclins on cyclin B1 promoter activation, we co-transfected cyclin A. cyclin E and cyclin D1 expressing plasmids with a cyclin B1 promoter construction driving a luciferase reporter gene into NIH 3T3 cells. All three cyclins produced activation of the reporter gene, however, co-transfection of cyclin A with a Cdk2 dominant negative mutant blocks activation by cyclin A. Our results suggest that cyclin B1 activation depends on Cdk2 kinase activity associated to cyclin A. To our knowledge this is the first report showing positive effect of cyclin A, cyclin E and cyclin D1 on cyclin B1 activation and blocking of this activation by a Cdk2 dominant negative mutant.
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PMID:Effect of cyclins and Cdks on the cyclin B1 promoter activation. 913 22

Transcription of the B-Myb gene is cell cycle regulated by an E2F-dependent mechanism and its product, B-Myb, is itself a transcription factor required for S-phase entry. Previously, we have shown that B-Myb is specifically phosphorylated during S-phase and that similar modification to a less electrophoretically mobile form could be induced by baculovirus-expressed cyclin A/Cdk2 kinase. We report here that cyclin A-mediated phosphorylation of B-Myb is associated with a marked increase in transactivation function in U-2 OS cells. In contrast to previous studies, transactivation of the luciferase reporter was dependent upon Myb binding sites located upstream of the promoter. Enhancement of B-Myb activation function was also obtained by truncation of the C-terminus just downstream of a domain conserved in evolution. Potentiation of B-Myb activity by phosphorylation was not simply a consequence of overcoming the negative effect of the C-terminus, however, as the truncated protein was to a lesser extent also activated by cyclin A/Cdk2. Whereas wild-type B-Myb transactivation activity could not be potentiated by cyclin A/Cdk2 in NIH3T3 cells, the truncated protein was hyperactive. Finally, we showed that B-Myb synergises with cyclin A to promote U-2 OS cells into S-phase.
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PMID:B-Myb function can be markedly enhanced by cyclin A-dependent kinase and protein truncation. 918 59


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