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

The orderly progression through the cell cycle is mediated by the sequential activation of several cyclin/cyclin-dependent kinase (cdk) complexes. These kinases phosphorylate a number of cellular substrates, among which is the product of the retinoblastoma gene, pRb. Phosphorylation of pRb in late G1 causes the release of the transcription factor E2F from pRb, resulting in the transcriptional activation of E2F-responsive genes. We show here that phosphorylation of the pRb-related p107 is also cell cycle regulated. p107 is first phosphorylated at 8 hr following serum stimulation of quiescent fibroblasts, which coincides with an increase in cyclin D1 protein levels. Consistent with this, we show that a cyclin D1/cdk4 complex, but not a cyclin E/cdk2 complex, can phosphorylate p107 in vivo. Furthermore, phosphorylation of p107 can be abolished by the overexpression of a dominant-negative form of cdk4. Phosphorylation of p107 results in the loss of the ability to associate with E2F-4, a transcription factor with growth-promoting and oncogenic activity. A p107-induced cell cycle block can be released by cyclin D1/cdk4 but not by cyclin E/cdk2. These data indicate that the activity of p107 is regulated by phosphorylation through D-type cyclins.
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PMID:Regulation of the retinoblastoma protein-related p107 by G1 cyclin complexes. 779 74

In quiescent cells, cdc2 mRNA is almost undetectable. Stimulation of cells to reenter the cell cycle results in induction of cdc2 expression, beginning at the G1-to-S transition and reaching maximum levels during late S and G2 phases. To investigate cdc2 transcriptional regulation throughout cell cycle progression, we monitored protein-DNA interactions by in vivo footprinting along 800 bp of the human cdc2 promoter in quiescent fibroblasts and at different time points following serum stimulation. We found 11 in vivo protein-binding sites, but no protein binding was observed at a high-affinity E2F site that had previously been implicated in cdc2 regulation. Nine of the identified in vivo binding sites (among them were two inverted CCAAT boxes, two Sp1 sites, and one ets-2 site) bind transcription factors constitutively throughout the cell cycle. However, at two elements located at positions -60 and -20 relative to the transcription start site, the binding pattern changes significantly as the cells are entering S phase. A G0- and G1-specific protein complex disappears at the -20 element at the beginning of S phase. This sequence deviates at one base position from known E2F consensus binding sites. We found that the major E2F activity in human fibroblasts contains E2F-4 and p130. The -20 element of the cdc2 gene specifically interacts with a subset of E2F-4-p130 complexes present in G0 cells but does not interact with S-phase-specific E2F complexes. Transient-transfection experiments with wild-type and mutant cdc2 promoter constructs indicate that the -20 element is involved in suppressing cdc2 activity in quiescent cells. We suggest that the presence of the p130-E2F-4 complex in G0/G1 blocks access of components of the basal transcription machinery or prevents transaction by the constitutively bound upstream activator proteins.
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PMID:In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression. 852 57

p21Sdi1/WAF1/Cip1 inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the phosphorylation of growth-regulatory proteins, including the retinoblastoma tumor suppressor protein (pRb). The ultimate effector(s) of p21 growth inhibition, however, is largely a matter of conjecture. We show that p21 inhibits the activity of E2F, an essential growth-stimulatory transcription factor that is negatively regulated by unphosphorylated pRb. p21 suppressed the activity of E2F-responsive promoters (dihydrofolate reductase and cdc2), but E2F-unresponsive promoters (c-fos and simian virus 40 early) were unaffected. Moreover, the simian virus 40 early promoter was rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 deletion mutants showed good agreement in their abilities to inhibit E2F transactivation and DNA synthesis; and E2F-1 (which binds pRb), but not E2F-4 (which does not), reversed both inhibitory effects of p21. Despite the central role for pRb in regulating E2F, p21 suppressed growth and E2F activity in cells lacking a functional pRb. Moreover, p21 protein (wild type but not mutant) specifically disrupted an E2F-cyclin-dependent protein kinase 2-p107 DNA binding complex in nuclear extracts of proliferating cells, whether or not they expressed normal pRb. Thus, E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for the inhibition of growth or E2F-dependent transcription.
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PMID:Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein. 864 10

During the transition from G1 to G0, p130 undergoes a specific phosphorylation event-leading to p130-form 2- that is mediated by a kinase/s other than the known G1, S and G2/M cyclin/CDKs. Changes in the phosphorylation status of p130 during this transition are responsible, at least in part, for the concomitant formation of p130/E2F-4 complexes, which are characteristic of G0. These complexes remain abundant during early G1 upon restimulation, but not after mitosis, and are dissociated in mid G1 when p130 is abruptly hyperphosphorylated to form 3. Subsequently, p130 forms 1 and 2 are no longer detected during the remainder of the cell cycle. Consistently, phosphorylation to form 3 and dissociation from E2F-4 complexes is reproduced by a cyclin/CDK holoenzyme in vitro. TGF-beta-induced G1 arrest abrogates cyclin/CDK phosphorylation of p130 but not phosphorylation to form 2. The cell cycle-dependent phosphorylation pattern of p130 is thus shown to comprise two distinct steps that are catalyzed by different kinases. The differential regulation of p130 and pRB phosphorylation during the transition from G1 to G0 may explain the fact that p130 and E2F-4 are the major components of E2F complexes in quiescent cells. Moreover, the newly described phosphorylation of p130 at the transition from G1 to G0 defines a novel mechanism of cell cycle exit regulation.
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PMID:G1 cyclin/CDK-independent phosphorylation and accumulation of p130 during the transition from G1 to G0 lead to its association with E2F-4. 871 Mar 62

B-myb belongs to a group of cell cycle genes whose transcription is repressed in G0/early G1 through a binding site for the transcription factor E2F. Here, we show that the B-myb repressor element is specifically recognised by heterodimers consisting of DP-1 and E2F-1, E2F-3 or E2F-4. Surprisingly, E2F-mediated repression is dependent on a contiguous corepressor element that resembles the CHR previously established as a corepressor of the CDE in cell cycle genes derepressed in S/G2, such as cyclin A, cdc2 and cdc25C. A factor binding to the B-myb CHR was identified in fractionated HeLa nuclear extract and found to interact with the minor groove, as previously shown by in vivo footprinting for the cyclin A CHR. The B-myb and cdc25C CHRs are related with respect to protein binding but are functionally clearly distinct. Our results support a model where both E2F- and CDE-mediated repression, acting at different stages in the cell cycle, are dependent on promoter-specific CHR elements.
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PMID:Cell cycle-regulated repression of B-myb transcription: cooperation of an E2F site with a contiguous corepressor element. 876 Aug 72

The cytodifferentiation agent hexamethylene bisacetamide (HMBA) is an inducer of differentiation of a variety of transformed cells, including the murine erythroleukemia (MEL) cell line. Induction of differentiation of MEL cells is a multistep process resulting in cessation of cell division and phenotypic maturation (including hemoglobin production). To investigate HMBA-induced MEL cell differentiation, we have analyzed the regulation of the E2F transcription factor. E2F regulates the transcription of several genes whose products are involved in both cell cycle regulation and differentiation. In nuclear extracts from uninduced MEL cells, three complexes were detected using gel mobility assays with the E2F/E2 oligonucleotide. The complex with the fastest mobility is the free form of E2F binding to DNA, and the more slowly migrating complexes contain E2F, p107, and cdk2. By 8 h of HMBA induction and for the remainder of the differentiation process, the free E2F complex is not detected, and only complexes of slower mobility, which contain p107 and cdk2, are found. The level of p107 protein increases during induction of differentiation; there is no change in the level of cdk2 protein and E2F-4 and DP-1 proteins during the first 4 days. The level of E2F-1 mRNA does not change, but a new form of E2F is detected during induction of differentiation. Thus, HMBA causes a selective loss in the free E2F DNA-binding complex, an increase in p107 protein, and an increase in a form of E2F protein during MEL cell differentiation.
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PMID:Changes in E2F DNA-binding activity during induced erythroid differentiation. 878 31

The cyclin-dependent kinases cdk2 and cdk3 are required for the G1-S transition in mammalian cells. Here we show that G1 arrest induced by the corresponding dominant-negative mutants of these enzymes, cdk2dn or cdk3dn, is resistant to the action of SV40 T antigen (T). In the presence of cdk2dn, T released active E2F from negative control by pRb and its related family members (pocket proteins) but failed to induce S-phase. Therefore, among other targets, cdk2 also phosphorylates nonpocket protein substrates in promoting S-phase entry, and T does not mimic all cdk2 functions. In the presence of cdk3dn, however, T failed to induce cell cycle progression or stimulate E2F-dependent transcription activity. Dominant-negative cdk3 inhibited E2F-1, E2F-2, and, less significantly, E2F-3, but not E2F-4 transcription activity. The inhibition occurred in a pRb-independent manner and did not affect the DNA-binding capacity of the transcription factor. Cdk3 bound specifically to E2F-1/DP-1 complexes in vivo, most likely through DP-1. Thus, cdk3 function contributes to the activation of E2F-1, E2F-2, and partially E2F-3 and, thereby, participates in the process of S-phase entry.
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PMID:Differential effects of cdk2 and cdk3 on the control of pRb and E2F function during G1 exit. 884 21

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

Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.
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PMID:MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally differentiated myocytes. 912 66

E2F is a heterodimeric transcription factor that controls transcription of several growth-regulatory genes including cdc2. To investigate the mechanism of interferon-alpha (IFN-alpha)-mediated growth suppression of hematopoietic cells, we examined the effect of IFN-alpha on the expression and function of E2F using IFN-sensitive Daudi cells. Down-regulation of E2F-1, a subunit of E2F, was observed after 8 h of culture with IFN-alpha; expression of E2F-4, another subunit of E2F, and DP-1, a heterodimeric partner of E2F, was unaffected. Gel shift assays revealed that the DNA binding activity of free E2F, which is composed of E2F-1 and E2F-4, was inhibited by IFN-alpha. In contrast, IFN-alpha did not affect the DNA binding ability of E2F-1 and E2F-4 in a complex with retinoblastoma (RB) susceptibility gene family proteins including pRB, p107, and p130. IFN-alpha could induce dephosphorylation of pRB, thereby turning active E2F-pRB complexes into transcriptional repressors. Transient chloramphenicol acetyltransferase assays revealed that the activity of the E2F-dependent cdc2 promoter was suppressed by IFN-alpha. These results suggest that the antiproliferative action of IFN-alpha is mediated through the modulation of E2F activity in two different ways: down-regulation of transcriptionally active free E2F and conversion of E2F-pRB complexes into transcriptional repressors.
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PMID:Modulation of E2F activity is linked to interferon-induced growth suppression of hematopoietic cells. 913 87


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