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)

Transcription of the murine thymidine kinase gene, which is coregulated with the G1/S phase transition, is activated by changing the binding of protein complexes Yi1 and Yi2 to three upstream DNA motifs. Yi1 is replaced by Yi2 shortly before S phase. Yi1 contains a protein of 110 kDa that binds to the DNA motif sites and may be an underphosphorylated murine retinoblastoma protein, shown by its molecular mass, timing of its activity, and antibody recognition. An H1 kinase related to cdc2 cofractionates with both complexes. We propose that this kinase phosphorylates the murine retinoblastoma protein, releasing transcriptional inhibitions by Yi1 and permitting cell cycle progression. These results provide a cycle-related molecular target for such complexes. They are based on investigations of cycle control in uninfected cells. The Yi complexes are similar but not identical to complexes that include a cellular protein, E2F, that was originally found to bind to adenovirus DNA.
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PMID:Thymidine kinase transcription is regulated at G1/S phase by a complex that contains retinoblastoma-like protein and a cdc2 kinase. 156 17

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.
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PMID:Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. 762 16

Proliferating cells characteristically undergo programmed (i.e. apoptotic) death if their progression through the cell cycle is sufficiently perturbed. To determine whether androgen ablation-induced programmed death of prostatic glandular cells involves apoptosis triggered by recruitment of nonproliferating cells into a perturbed cell cycle, rat ventral prostates were assessed temporally after castration for several stereotypical molecular stigmata of entry into the proliferative cell cycle. Northern blot analysis was used to assess levels of transcripts from genes characteristically activated 1) during the transition from quiescence (G(0)) into G1 of the proliferative cell cycle (cyclin-D1 and cyclin-C), 2) during the transition from G1 to S (cyclin-E, cdk2, thymidine kinase, and H4-histone), and 3) during progression through S (cyclin-A). Although levels of each of these transcripts increased as expected in prostatic glandular epithelial cells stimulated to proliferate by the administration of exogenous androgen to previously castrated rats, levels of the same transcripts decreased in prostatic glandular cells induced to undergo apoptosis after androgen withdrawal. Northern and Western blot analyses also demonstrated that there was no increase in prostatic p53 messenger RNA or protein content per cell after androgen ablation. Likewise, after castration, there was no enhanced prostatic expression of the WAF1/CIP1 gene, a gene whose expression is known to be induced in both a p53-dependent and -independent manner during recruitment from G0 into G1. In addition, androgen ablation-induced apoptosis of prostatic glandular cells was not accompanied by retinoblastoma protein phosphorylation, which is characteristic of progression into late G1. Nuclear run-on assays demonstrated that there was no increase in the prostatic rate of transcription of the c-myc and c-fos genes after castration. These results demonstrate that prostatic glandular cells undergo programmed death in G(0) without recruitment into the G1 phase of a defective cell cycle, and that an increase in p53 protein or its function is not involved in this death process.
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PMID:Androgen ablation-induced programmed death of prostatic glandular cells does not involve recruitment into a defective cell cycle or p53 induction. 772 Jun 36

Glucocorticoids inhibit transcription of the proto-oncogene c-myc in lymphoid cells of thymic origin. To determine if this effect is associated with changes in the properties of the transcription factor E2F, extracts were prepared from control and glucocorticoid-treated P1798 murine T lymphoma cells, and the macromolecular state of E2F was assessed by gel-mobility shift. Control extracts exhibit two predominant gel-mobility shift entities of which one corresponds to "free" E2F. A second entity, complex C, has properties similar to those described for the complex containing E2F, p107, cyclin A, and Cdk2. Complex C disappears after addition of dexamethasone and is replaced by complex D. The mobility of this complex and its sensitivity to SV40 T antigen suggest that complex D corresponds to an E2F-p105Rb-1 complex. Extracts from control and glucocorticoid-treated cells yield identical DNase I protection patterns on the c-myc P2 promoter. Furthermore, such extracts transcribe the c-myc P2 promoter in vitro with equal activity. The relative abundance of the E2F complexes was measured after addition of dexamethasone. Complex C disappears as cells withdraw from S phase, and complex D appears at this time. The genes encoding thymidine kinase (Tk-1) and p34cdc2 (cdc2) are regulated with kinetics similar to those observed for changes in the macromolecular state of E2F. However, regulation of c-myc expression occurs long before any change in E2F. The macromolecular state of E2F may regulate expression of genes at the G1/S boundary. However, the data are not consistent with the hypothesis that association of E2F with tumor suppressor gene products such as p107 or p105Rb-1 is relevant to glucocorticoid regulation of c-myc transcription.
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PMID:The macromolecular state of the transcription factor E2F and glucocorticoid regulation of c-myc transcription. 800 8

By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
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PMID:G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. 828 95

Promoter elements that are important for the G1-S induction of the human thymidine kinase (htk) promoter reside within the core of the cell cycle regulatory unit, positioned between -110 and -84 upstream of the TATA element. Within this 27-bp region are three GC-rich motifs, which resemble the E2F binding site. By site-directed mutagenesis, we identified a 14-bp region, between -97 and -84, critical for the htk promoter transcriptional activity. Methylation interference studies indicate that the sequences between -97 and -84 are major protein contact points, correlating with the functional significance of this sequence in vivo. Although the core of the cell cycle regulatory unit contains three E2F-like sites and can form minor S-phase-specific complexes containing p107, cyclin A, and cdk2, the major complex that binds to this region is not competed by E2F binding sites. Through DNA affinity chromatography, we identified a set of protein species of approximately 40 kDa that copurified with the htk DNA binding activity. From gel shift assays and Western blot analysis, this protein species is antigenically distinct from E2F-1, E2F-2, E2F-3, and E2F-4. Our studies raise the possibility that other members of the E2F protein family or a novel protein(s) with preferred binding affinity for the htk promoter exert(s) control on the G1 to S regulation of the htk promoter through their interactions with cyclins and kinases.
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PMID:Identification of a set of protein species approximately 40 kDa as high-affinity DNA binding factor(s) to the cell cycle regulatory region of the human thymidine kinase promoter. 895 43

Small DNA viruses (adenoviruses, simian virus 40, or human papillomaviruses) induce S-phase progression but prevent cell division to provide precursors for viral DNA replication. Herpes simplex viruses types 1 or 2 (HSV-1 or HSV-2) contain genes which encode DNA-metabolizing enzymes, for example, ribonucleotide reductase, thymidine kinase and dUTPase, suggesting that S-phase factors are not required for an efficient infection. However, several studies indicated that HSV induces some events that occur during cell-cycle progression. To determine if HSV-2 induces S-phase entry, we examined serum-arrested African green monkey kidney cells (CV-1) after infection. Two hours after infection steady-state levels of the S-phase-specific cyclin, cyclin A, increased. S-phase cyclin-dependent kinase activity (CDK2) was stimulated 10-fold 8 h after infection but decreased at 16 or 24 h after infection. Mitotic CDK activity (CDC2) was not activated after infection, in part due to decreases in CDC2 protein levels and inactivation of enzymatic activity resulting from tyrosine phosphorylation of CDC2. Furthermore, CDK4 activity was not dramatically affected by infection. These studies indicate that HSV-2 infection selectively activates CDK2 after infection but cell-cycle progression does not occur. We hypothesize that infection activates certain components of the cell cycle which enhance viral gene expression and DNA replication.
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PMID:Analysis of cyclin-dependent kinase activity after herpes simplex virus type 2 infection. 940 Sep 86

It has been reported that the polypeptide of thymidine kinase type 1 (TK1) from human and mouse cells can be modified by phosphorylation. Our laboratory has further shown that the level of human TK phosphorylation increases during mitotic arrest in different cell types (Chang, Z.-F., Huang, D.-Y., and Hsue, N.-C. (1994) J. Biol. Chem. 269:21249-21254). In the present study, we demonstrated that a mutation converting Ser13 to Ala abolished the mitotic phosphorylation of native TK1 expressed in Ltk- cells. Furthermore, we expressed recombinant proteins of wild-type and mutated human TK1 with fused FLAG epitope in HeLa cells, and confirmed the occurrence of mitotic phosphorylation on Ser13 of hTK1. By using an in vitro phosphorylation assay, it was shown that wild-type hTK1, but not mutant TK1(Ala13), could serve as a good substrate for Cdc2 or Cdk2 kinase. Coexpression of p21(waf1/cip1), which is a universal inhibitor of Cdk kinases, in Ltk- fibroblasts also suppressed mitotic phosphorylation of hTK1 expressed in this cell line. Thus, Cdc2 or related kinase(s) is probably involved in mitotic phosphorylation on Ser13 of the hTK1 polypeptide. We also found that mutation on Ser13 did not affect the functional activity of hTK1. As the sequences around Ser13 are highly conserved in vertebrate TK1s, we speculate that phosphorylation of Ser13 may play a role in the regulation of TK1 expression in the cell cycle.
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PMID:Serine 13 is the site of mitotic phosphorylation of human thymidine kinase. 957 53

Confluent 3T3-L1 preadipocytes differentiate to adipocytes in the presence of insulin, dexamethasone, and isobutylmethylxanthine (IDI). A transient increase of DNA synthesis is induced in 3T3-L1 cells 18 h after addition of IDI, followed by an arrest in the G1 phase of the cell cycle. Growth arrested cells express the proto-oncogene c-myc and the gene for the CCAAT/enhancer binding protein (C/EBPalpha) between day 2 and 5. While c-Myc is strongly implicated in cell proliferation, C/EBPalpha: is a differentiation-specific transcription factor with antiproliferative activity. Here we have characterized the cell cycle arrest in differentiating 3T3-L1 cells. Arrested cells express the Cdk inhibitors p21 and p27, but, at the same time, show hyperphosphorylation of Rb and expression of the E2F-regulated thymidine kinase gene. The addition of new serum to arrested cells resulted in cyclin A expression and Cdk2 activity, but not in DNA synthesis. Simian virus 40 large tumor antigen (LTAg) is a potent mitogen. The mutant LTAg-K1, deficient in binding of pocket proteins and unable to induce DNA synthesis in serum-starved 3T3-L1 cells, efficiently induced DNA synthesis in differentiating 3T3-L1 cells. This indicates that pocket proteins are probably not involved in the control of the cell cycle arrest during 3T3-L1 cell differentiation. Our data suggest that the differentiation-specific cell cycle block in 3T3-L1 cells is resistant to high levels of c-Myc, inactivation of pocket proteins, upregulation of cyclin A levels, and Cdk2 activation, but can be abolished by a function of LTAg that is independent of binding to pocket proteins.
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PMID:Analysis of cell cycle arrest in adipocyte differentiation. 992 2

Our previous studies have shown that treatment of MCF-7 breast cancer cells with cytokine oncostatin M (OM) results in a growth arrest and a concurrent decrease in p53 expression. It remains to be determined whether these two important events are directly connected, as changes in p53 protein levels can lead to variable biological outcomes. In this study we have generated stable cell lines (MCF7-ptsp53) that express p53Val135 a p53 temperature-sensitive mutant. We demonstrate that overexpression of the wildtype (wt) p53 at permissive temperature in MCF7-ptsp53 cells leads to growth arrest at the G2-M phase of the cell cycle. Inhibition of endogenous p53 function with the expression of mutant p53 protein at non-permissive temperature did not affect the OM-induced G1 cell cycle arrest. Microarray studies were further carried out to identify p53- and OM-regulated genes that mediate the G2/M or G1 cell cycle arrest. We show that the expression of p21 was upregulated and expressions of cdc2, cyclin B2 and protein regulator of cytokinesis 1 (PRC1) were suppressed by overexpression of the wt p53 in MCF7-ptsp53 cells at the permissive temperature. In contrast, OM treatment caused coordinate changes of mRNA expression of several cell cycle components including c/EBPdelta, cdc20, and thymidine kinase 1 (TK1) that mainly affect G1-S phase transition. All together, our results suggest that the downregulation of p53 transcription may be involved in some other cellular changes induced by OM but it is not directly connected to the antiproliferative activity of OM per se.
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PMID:Molecular characterization of oncostatin M-induced growth arrest of MCF-7 cells expressing a temperature-sensitive mutant of p53. 1288 96


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