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)

Transcription of the human cdc2 gene is cell cycle regulated and restricted to proliferating cells. Nuclear run-on assays show that cdc2 transcription is high in S and G2 phases of the cell cycle but low in G1. To investigate transcriptional control further, genomic clones of the human cdc2 gene containing 5' flanking sequences were isolated and shown to function as a growth regulated promoter in vivo when fused to a CAT reporter gene. In primary human fibroblasts, the human cdc2 promoter is negatively regulated by arrest of cell growth in a similar fashion to the endogenous gene. This requires specific 5' flanking upstream negative control (UNC) sequences which mediate repression. The retinoblastoma susceptibility gene product (Rb) specifically represses cdc2 transcription in cycling cells via 136 bp of 5' flanking sequence located between -245 and -109 within the UNC region. E2F binding sites in this region were shown to be essential for optimal repression. A model is proposed where Rb negatively regulates the cdc2 promoter in non-cycling and cycling G1 cells.
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PMID:Cell cycle regulation of the human cdc2 gene. 158 9

The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc- phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34cdc2 contains no phosphoserine.
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PMID:Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase. 203 6

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

Members of the recently discovered family of cyclin-dependent kinases inhibitors (CKIs) appear to play an essential regulatory role in the control of cell proliferation. To investigate the molecular basis of the interaction between these proteins and the cyclin-dependent kinases (CDKs), we performed a systematic mutagenesis of the CKI family member p21Cip1 using the alanine-scanning strategy. We have examined the interaction between in vitro translated human cdk2, cyclins A and D1, purified proliferating cell nuclear antigen (PCNA) and a set of human p21Cip1 mutants fused to glutathione S-transferase. Independent domains that are required for the interaction with cdk2 and with PCNA have been identified. The cdk2 binding domain is located in the N-terminal part of the protein, between residues 45 and 60, a region that is fully conserved in the p27Kip1 inhibitor. A PCNA binding region was localised to the C-terminus of the protein, between residues 142 and 163. These findings define protein motifs that are highly conserved between members of the CKI family and that are likely to play an essential function in the regulation of the G1/S transition.
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PMID:Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. 778 76

CDK2 (cyclin-dependent kinase 2) is a serine/threonine kinase which is involved in regulating S-phase entry in higher eukaryotes. To investigate the transcriptional control of this gene, a 13-kb Xenopus laevis genomic clone containing the 5' flanking sequences was isolated. A 2.7-kb fragment containing the promoter region was sequenced and the transcription start point (tsp) was determined by primer extension. Several putative regulatory elements, such as the E2F-binding site, Y box and octamer-binding site, were localized in this region, but no TATA box was found. When fused to cat, a reporter gene encoding chloramphenicol acetyltransferase, the 5' flanking sequences were shown to function in oocytes and an enhancer activity was found in this region. During early embryogenesis, cdk2 promoter activity was tested and de novo transcription was detected at the mid-blastula transition.
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PMID:Cloning of the Xenopus laevis cdk2 promoter and functional analysis in oocytes and during early development. 782 9

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.
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PMID:Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development. 806 20

Terminally differentiated skeletal muscle myotubes are arrested in G0 phase of the cell cycle and are unable to be released from this arrest by stimulation with mitogens including serum and growth factors. To inspect a possibility of reversing the quiescence at the G0 phase, we have exploited the mouse skeletal muscle cell line C2SVTts11, which is a clone of C2 cells transfected with the SV40 T antigen gene (encoding thermolabile large T and wild-type small t) fused to an inducible promoter. When the large T is induced in the myotubes, the terminally differentiated cells reenter the cell cycle and proceed to S and M phases. To elucidate how large T forces the myotubes to traverse each phase of the cell cycle, we examined the expression and activity of Cdk2 and Cdc2, which in complex with cyclin A and cyclin B are essential for S and M phases, respectively in undifferentiated cells. The levels of their mRNAs and proteins and histone H1 kinase activity, which was ascribed to Cdc2-cyclin B, were high in the proliferating myoblasts but gradually decreased during terminal differentiation. In contrast, they were reinduced in the myotubes reentering the cell cycle. Stimulation of the myotubes with serum failed to evoke these factors. These results indicate that large T, but not mitogens, is able to drive terminally differentiated myotubes to pass each phase of the cell cycle through eliciting these factors as do mitogens on proliferating undifferentiated cells. Since large T is a nuclear protein, signals generated by the protein in the nucleus are likely to be sufficient to induce each phase of the cell cycle in the terminally differentiated cells.
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PMID:SV40 large T antigen reinduces the cell cycle in terminally differentiated myotubes through inducing Cdk2, Cdc2, and their partner cyclins. 808 30

The amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.
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PMID:Isolation of two novel cDNAs whose products associate with the amino terminus of the E2F1 transcription factor. 882 66

We tested the ability of synthetic peptides derived from p21(WAF1), fused to the internalization peptide sequence derived from Antennapedia, to inhibit the growth of cancer cells in two human ovarian cancer cell lines expressing wild-type p53 or not. Two fused peptides corresponding to p21(WAF1) regions 17-33 and 63-77 inhibited cell growth in both cell lines while the same peptides without the internalization sequence were inactive. The fused peptides prevented growth at concentrations which inhibited cyclin-dependent kinase 2 and cdc2 activity, thus demonstrating that the peptides act by mimicking the action of p21(WAF1) on kinases. This study illustrates the potential pharmacological use of small peptides fused with the Antennapedia internalization sequence in proliferative disorders. The approach may be extended to other diseases in which cell penetration of a peptide may be of therapeutic benefit. More stable drug-like molecules with better pharmacological properties could be designed based on the results obtained with peptides.
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PMID:p21WAF1-derived peptides linked to an internalization peptide inhibit human cancer cell growth. 910 43

Nucleolin is a major component of the nucleolus. In Xenopus laevis, a maternal store of nucleolin is accumulated in the multiple nucleoli generated during oogenesis. This maternal nucleolin is distributed throughout the cytoplasm of the egg during oocyte maturation and after fertilization it is gradually reaccumulated in the nuclei of the embryo. Cytoplasmic localization of nucleolin coincides with massive phosphorylation by p34cdc2 kinase, and nuclear translocation is accompanied by net dephosphorylation. Multiple phosphorylation consensus sites for the cell cycle-dependent p34cdc2 kinase and for protein kinase CK2 are clustered in the N-terminal domain of nucleolin. To assess the efficiency of the bipartite nuclear localization signal, we have constructed fusion proteins consisting of maltose binding protein (MBP) and the nuclear localization signal of nucleolin. In addition, either an acidic domain of nucleolin without phosphorylation sites, or an acidic domain containing 4 CK2 sites, or a cluster of 5 cdc2 sites was fused to the MBP-nuclear localization signal (MBP-NLS). Nuclear translocation of these constructs was tested in an in vitro system consisting of Xenopus egg extract and sperm nuclei. Nuclear targetting of MBP by the bipartite nuclear localization signal of nucleolin became significantly more efficient after addition of either CK2 sites or cdc2 sites to the MBP-NLS construct. Yet the cdc2 sites play a dual role. They enhance nuclear translocation exclusively in their dephosphorylated state and promote cytoplasmic localization when phosphorylated, thereby providing a powerful cell cycle-dependent regulatory element of the nuclear localization signal.
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PMID:Protein phosphorylation sites regulate the function of the bipartite NLS of nucleolin. 927 Aug 71


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