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)

It is a characteristic of the central nervous system of higher eukaryotes that neurons, after an initial proliferation phase, remain postmitotic for their whole life span. In the developing quail neuroretina, most retinoblasts become postmitotic after 7-8 days of incubation. They also cease to express cdc2, which is presumably necessary to allow retinoblasts to definitively leave the cell cycle. The molecular mechanisms monitoring cdc2 expression during differentiation remain partly understood. To further study the control of cdc2 transcription in avian cells, we have cloned the quail cdc2 promoter. Two functional regulatory elements have been characterized. One of them contains an E2F-binding site. Human E2F-1 was found to transactivate the quail cdc2 promoter very efficiently in avian and human cells. Gel retardation experiments are presented, suggesting that E2F, in association with different partners, is a major regulatory of cdc2 transcription during the development of the neuroretina. Our data also indicate that another transcription factor binds to the octamer CAGGTGGC located 115 nucleotides above the main transcription start site. This motif is thus another important regulatory element participating in the control of cdc2 expression.
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PMID:Two distinct regulatory elements control quail cdc2 transcription: possible involvement in the control of retinoblast differentiation. 883 64

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

A spatially well organized continuum of proliferation, differentiation, and death is displayed along crypt-villus units in the adult mouse small intestine. This continuum provides an opportunity to examine in vivo the mechanisms by which proliferative status changes as a function of cellular differentiation. Immunohistochemical studies of normal FVB/N mice revealed that as epithelial cells complete their terminal differentiation during a 48-72-h migration up villi, there is a marked and rapid fall in the levels of two important regulators of the G1/S transition, cyclin D1 and cyclin-dependent kinase (cdk) 2. However, cellular levels of their partners, cdk4 and cyclin E, remain unchanged as does the level of pRB. Adult FVB/N transgenic mice were studied that contained an intestinal fatty acid binding protein gene promoter (Fabpi) linked to wild type Simian virus 40 large T antigen (SV40 TAgWt) or a mutant TAg with Lys for Glu substitutions at residues 107 and 108 (SV40 TAgK107/8) that fails to bind pRB and related pocket proteins. Both transgenes are expressed only in villus enterocytes. SV40 TAgWt causes these terminally differentiated cells to re-enter the cycle. Re-entry is accompanied by a reduction in un/hypophosphorylated pRB, an induction of cyclin D1 and cdk2, but no change in cdk4, cyclin E, or E2F-1. In contrast, SV40 TAgK107/8 fails to induce re-entry and does not produce changes in un/hypophosphorylated pRB, cyclin D1, or cdk2 accumulation. These results suggest that un/hypophosphorylated pRB is an important mediator of the cell cycle arrest that normally occurs as enterocytes exit the crypt and complete their differentiation. Fabpi-directed expression of E2F-1 does not cause villus enterocytes to return to the cell cycle, alter their suppression of cyclin D1 or cdk2, or affect their state of differentiation, emphasizing the insensitivity of these cells to the effects of E2F-1. Analyses of p53(-/-) and p53(+/+) mice containing Fabpi-SV40 TAgWt and Fabpi-SV40 TAgK107/8 established that the proliferation induced by SV40 TAgWt does not require p53 and is associated with increased (p53-independent) apoptosis. The presence of cyclin E and cdk4 in differentiating villus enterocytes emphasizes that these cells retain part of their proliferative heritage expressed 24-72 h earlier in the crypt. The data suggest that down-regulation of cdk2 and/or cyclin D1 expression may be important for control of proliferative status and/or execution of terminal differentiation.
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PMID:Use of normal and transgenic mice to examine the relationship between terminal differentiation of intestinal epithelial cells and accumulation of their cell cycle regulators. 891 Apr 66

Normal somatic cells of higher organisms do not divide indefinitely. After a finite number of divisions, normal cells irreversibly cease proliferation by a process termed replicative or cellular senescence. Replicative senescence is controlled by multiple, dominant-acting genes about which very little is known. The only genes known to reactivate DNA synthesis in senescent cells are viral oncogenes encoding proteins that bind and inactivate the p53 and retinoblastoma (pRb) tumor suppressor proteins. SV40 T antigen is the best studied of these viral oncoproteins. T[K1] is a T antigen point mutant that selectively is defective in binding pRb and the pRb-related proteins p107 and p130. We show that T[K1] stimulated quiescent human fibroblasts to synthesize DNA nearly as well as wild-type T but was incapable of stimulating senescent cells. We tested several growth regulatory genes that are repressed in senescent cells for ability to restore activity to T[K1]. These included c-fos, c-jun, Id-1, Id-2, E2F-1, and cdc2. Only the helix-loop-helix (HLH) protein, Id-1, restored the ability of T[K1] to reactivate DNA synthesis in senescent cells. This activity of Id-1 was not shared by Id-2, a related protein, and depended on an intact HLH domain. It did not appear that Id-1 interacted directly with pRb or p107. Constitutive Id-1 expression failed to rescue proliferating cells from growth inhibition by pRb, p107, or p130, and failed to interact with pRb in the yeast two hybrid system. Because Id proteins negatively regulate basic-HLH (bHLH) transcription factors, we suggest that senescent cells express one or more bHLH factor that cooperates with pRb, or pRb-related proteins, to suppress proliferation.
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PMID:The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts. 893 78

E2F-1 plays a crucial role in the regulation of cell-cycle progression at the G1-S transition. In keeping with the fact that, when overproduced, it is both an oncoprotein and a potent inducer of apoptosis, its transcriptional activity is subject to multiple controls. Among them are binding by the retinoblastoma gene product (pRb), activation by cdk3, and S-phase-dependent down-regulation of DNA-binding capacity by cyclin A-dependent kinase. Here we report that E2F-1 is actively degraded by the ubiquitin-proteasome pathway. Efficient degradation depends on the availability of selected E2F-1 sequences. Unphosphorylated pRb stabilized E2F-1, protecting it from in vivo degradation. pRb-mediated stabilization was not an indirect consequence of G1 arrest, but rather depended on the ability of pRb to interact physically with E2F-1. Thus, in addition to binding E2F-1 and transforming it into a transcriptional repressor, pRb has another function, protection of E2F-1 from efficient degradation during a period when pRb/E2F complex formation is essential to regulating the cell cycle. In addition, there may be a specific mechanism for limiting free E2F-1 levels, failure of which could compromise cell survival and/or homeostasis.
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PMID:The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin-proteasome pathway. 895 96

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

Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS780PIPHIPR that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser780 in pRB was phosphorylated in the G1 phase in a cell cycle-dependent manner. Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo.
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PMID:The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2. 900 81

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

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.
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PMID:Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity. 918 31

Vitamin E succinate (VES) inhibited the proliferation of the estrogen receptor-negative human breast cancer cell line, BT-20, in the G1 phase of the cell cycle. The E2F proteins are integral transcriptional components in the regulation of cell growth. Overexpression of E2F-1 blocked the ability of VES to inhibit BT-20 cell growth, suggesting that VES regulation of E2F-1 activity leads to growth arrest of BT-20 cells. VES, although having little effect on E2F-1 steady-state protein levels, decreased E2F-1 phosphorylation and transactivation activity and increased cyclin A binding to E2F-1. GAL4-E2F-1 deletion mutant studies indicated that cyclin A negatively regulates E2F function. In VES-treated BT-20 cells, the cyclin A protein exhibited reduced kinase activity, which correlated with decreased steady-state levels and binding of cyclin-dependent kinase-2 to cyclin A and increased steady-state levels and binding of p21cip1 to cyclin A and cyclin-dependent kinase-2. The functional consequence of the negative regulatory effect of VES on E2F-1 function was shown by the ability of VES to inhibit the transcriptional activation of an E2F-1 responsive gene, c-myc. These studies show that VES induces growth inhibition of BT-20 cells through a mechanism that involves cyclin A-negative regulation of E2F-mediated transcription.
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PMID:Vitamin E succinate inhibits proliferation of BT-20 human breast cancer cells: increased binding of cyclin A negatively regulates E2F transactivation activity. 920 75


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