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 pocket domain of pRB is required for pRB to arrest the cell cycle. This domain was originally defined as the region of the protein that is necessary and sufficient for pRB's interaction with adenovirus E1A and simian virus s40 large T antigen. These oncoproteins, and other pRB-binding proteins that are encoded by a variety of plant and animal viruses, use a conserved LXCXE motif to interact with pRB. Similar sequences have been identified in multiple cellular pRB-binding proteins, suggesting that the viruses have evolved to target a highly conserved binding site of pRB that is critical for its function. Here we have constructed a panel of pRB mutants in which conserved amino acids that are predicted to make close contacts with an LXCXE peptide were altered. Despite the conservation of the LXCXE binding site throughout evolution, pRB mutants that lack this site are able to induce a cell cycle arrest in a pRB-deficient tumor cell line. This G(1) arrest is overcome by cyclin D-cdk4 complexes but is resistant to inactivation by E7. Consequently, mutants lacking the LXCXE binding site were able to induce a G(1) arrest in HeLa cells despite the expression of HPV-18 E7. pRB mutants lacking the LXCXE binding site are defective in binding to adenovirus E1A and human papillomavirus type 16 E7 protein but exhibit wild-type binding to E2F or DP, and they retain the ability to interact with CtIP and HDAC1, two transcriptional corepressors that contain LXCXE-like sequences. Consistent with these observations, the pRB mutants are able to actively repress transcription. These observations suggest that viral oncoproteins depend on the LXCXE-binding site of pRB for interaction to a far greater extent than cellular proteins that are critical for cell cycle arrest or transcriptional repression. Mutation of this binding site allows pRB to function as a cell cycle regulator while being resistant to inactivation by viral oncoproteins.
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PMID:Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins. 1077 61

By utilizing a human cDNA expression array blot (588 genes), we have observed overexpression of various transcription factors, cell cycle regulated kinases, and DNA repair genes in HTLV-1-infected T cells. One of the genes of interest, and focus in this study, is the cyclin-dependent kinase inhibitor, p21/waf1. The p21/waf1 transcription and protein is overexpressed in all HTLV-1-infected cell lines tested as well as ATL and HAM/TSP patient samples. While p21/waf1 has been shown to display a selectivity for G(1)/S cyclin/cdk complexes, we have observed p21/waf1 to be complexed with cyclin A/cdk2. Functionally, the association of p21/cyclin A/cdk2 decreased the histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, as well as affecting other substrates such as the C-terminus of Rb protein involved in c-Abl and HDAC1 regulation. Wild-type, but not a mutant form (M47) of Tax, was found to be able to transactivate the p21/waf1 promoter in a p53-independent manner. We found that the minimal p21/waf1 promoter (-49 to +49 sequence) was activated by Tax and the minimal promoter contained two E2A transcription factor binding sites located between the TATA box and the initiation site. E2A proteins, E12 and E47, as well as a related helix-loop-helix protein, HEB, are all up-regulated in HTLV-1-infected T cells. When using band shift analysis, we found that only the E1 site (overlapping the transcription start site) was a functional DNA binding site. By using a chromatin immunoprecipitation (ChIP) assay, we observed that histone H4, and not histone H3, was acetylated from the endogenous p21/waf1 promoter in vivo, implying that CBP/p300, and not the SAGA complex, was critical in complexing with E2A in up-regulation of p21/waf1 in HTLV-1-infected cells.
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PMID:Gene expression array of HTLV type 1-infected T cells: Up-regulation of transcription factors and cell cycle genes. 1108 Aug 12

HDAC1, a member of the histone deacetylase family, is involved in transcription regulation through the modification of chromatin structure. Several studies also implicated HDAC1 in tumorigenesis. Much attention has been concentrated on protein-protein interactions involving HDAC1 and the possibility that posttranslational modifications may occur in mammalian HDAC1 proteins has not been carefully and systematically investigated. In this study, we utilized in vivo labeling assays to demonstrate that both human and murine HDAC1 proteins are phosphorylated in cells. Assays using HDAC1 deletion mutants indicated that phosphorylation occurs in its C-terminal domain. cAMP-dependent kinase and casein kinase II, but not protein kinase C, cdc2, or MAP kinase, could phosphorylate HDAC1 in vitro, although HDAC1 contains several protein kinase C consensus sites. We also found that phosphorylation did not influence HDAC1 enzymatic activity using a human histone H4 N-terminal peptide as the substrate. Interestingly, HDAC1-FLAG fusion protein immunoprecipitated from transfected cells was found to be in association with a kinase activity, providing an in vitro assay for further studies of this posttranslational modification.
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PMID:Mammalian histone deacetylase 1 protein is posttranslationally modified by phosphorylation. 1132 22

Epigenetic silencing of the cell cycle proteins is one of the major pathways of the inhibition of liver proliferation in old mice. We recently identified glycogen synthase 3beta, GSK3beta, as an enzyme which controls the epigenetic regulation of the liver proliferation via reduction of cyclin D3-cdk4. The elevation of cyclin D3 in livers of old mice leads to the accumulation of a transcriptional C/EBPalpha-HDAC1-Brm complex and translational CUGBP1-eIF2 complex. The accumulation of the CUGBP1-eIF2 increases translation of HDAC1 and C/EBPbeta in livers of old mice leading to formation of C/EBPbeta-HDAC1 complex which represses C/EBPbeta-dependent genes by interacting with their promoters. The translational CUGBP1-eIF2 complex also plays critical role in the regulation of biological processes in young livers and needs to be tightly regulated. Our recent paper showed that this complex is un-appropriately upregulated in skeletal muscle precursors of patients affected with multisystemic disease, Myotonic Dystrophy 2 (DM2) leading to translational elevation of several proteins. These data suggest that the age-specific translational dysfunctions might be involved in DM2 pathogenesis. Our data also suggest that the regulation of translation is conserved in liver and skeletal muscle. In this review, we discuss biological consequences of the age-associated alterations of translation in the liver and a possible role of the elevation of the CUGBP1-eIF2 complex in development of DM2 pathology.
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PMID:GSK3beta-cyclin D3-CUGBP1-eIF2 pathway in aging and in myotonic dystrophy. 1957 75

The herpes simplex virus 1 ICP0 is a regulatory protein. Early in infection ICP0 localizes in ND10 bodies and performs two functions: As an E3 ligase in conjunction with E2 UbcH5a conjugating enzyme, it degrades the ND10 components PML and SP100. Concurrently, it suppresses the silencing of viral DNA by dispersing the HDAC1/CoREST/REST/LSD1 repressor complex. Subsequently, ICP0 is exported to the cytoplasm. In cells treated with HDAC inhibitors or transfected with irrelevant DNA, the export is delayed in a DNA dose-dependent fashion. Here, we follow up an observation that ICP0 binds cyclin D3 and that ICP0 mutants unable to bind cyclin D3 are not exported. Moreover, in infected cells cdk4 is activated, but cdk2 is not. We report that (i) cyclin D1, D2, or D3 colocalize with ND10 bodies and ICP0 early in infection and ultimately become incorporated into viral replication compartments, (ii) each of the D cyclins partially rescues DeltaICP0 mutants, and (iii) inhibition of cdk4 by inhibitor I sequesters ICP0 in the nucleus. A key finding is that overexpression of cyclin D3 enables the transport of ICP0 to the cytoplasm. We conclude that (i) ICP0 facilitates the recruitment of cyclin D3 to the sites of viral DNA synthesis, (ii) until its functions are completed, ICP0 is retained in the nucleus, and (iii) a common signal that results in the export of ICP0 to the cytoplasm is the accumulation of a viral DNA-synthesis-dependent late protein.
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PMID:ICP0 enables and monitors the function of D cyclins in herpes simplex virus 1 infected cells. 1970 44

Histone deacetylases (HDACs) regulate gene expression by deacetylating histones and also modulate the acetylation of a number of nonhistone proteins, thus impinging on various cellular processes. Here, we analyzed the major class I enzymes HDAC1 and HDAC2 in primary mouse fibroblasts and in the B-cell lineage. Fibroblasts lacking both enzymes fail to proliferate in culture and exhibit a strong cell cycle block in the G1 phase that is associated with up-regulation of the CDK inhibitors p21(WAF1/CIP1) and p57(Kip2) and of the corresponding mRNAs. This regulation is direct, as in wild-type cells HDAC1 and HDAC2 are bound to the promoter regions of the p21 and p57 genes. Furthermore, analysis of the transcriptome and of histone modifications in mutant cells demonstrated that HDAC1 and HDAC2 have only partly overlapping roles. Next, we eliminated HDAC1 and HDAC2 in the B cells of conditionally targeted mice. We found that B-cell development strictly requires the presence of at least one of these enzymes: When both enzymes are ablated, B-cell development is blocked at an early stage, and the rare remaining pre-B cells show a block in G1 accompanied by the induction of apoptosis. In contrast, elimination of HDAC1 and HDAC2 in mature resting B cells has no negative impact, unless these cells are induced to proliferate. These results indicate that HDAC1 and HDAC2, by normally repressing the expression of p21 and p57, regulate the G1-to-S-phase transition of the cell cycle.
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PMID:Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression. 2019 38

RUNX1 regulates formation of the definitive hematopoietic stem cell and its subsequent lineage maturation, and mutations of RUNX1 contribute to leukemic transformation. Phosphorylation of Ser-48, Ser-303, and Ser-424 by cyclin-dependent kinases (cdks) increases RUNX1 trans-activation activity without perturbing p300 interaction. We now find that endogenous RUNX1 interacts with endogenous HDAC1 or HDAC3. Mutation of the three RUNX1 serines to aspartic acid reduces co-immunoprecipitation with HDAC1 or HDAC3 when expressed in 293T cells; mutation of these three serines to alanine increases HDAC interaction, and mutation of each serine individually to aspartic acid also reduces these interactions. GST-RUNX1 isolated from bacterial extracts bound in vitro translated HDAC1 or HDAC3, and these interactions were weakened by mutation of Ser-48, Ser-303, and Ser-424 to aspartic acid. The ability of RUNX1 phosphorylation and not only serine to aspartic acid conversion to reduce HDAC1 binding was demonstrated using wild-type GST-RUNX1 phosphorylated in vitro using cdk1/cyclinB and by exposure of 293T cells transduced with RUNX1 and HDAC1 to roscovitine, a cdk inhibitor. Finally, RUNX1 or RUNX1(tripleD), in which Ser-48, Ser-303, and Ser-424 are mutated to aspartic acid, stimulated proliferation of transduced, lineage-negative murine marrow progenitors more potently than did RUNX1(tripleA), in which these serines are mutated to alanine, suggesting that stimulation of RUNX1 trans-activation by cdk-mediated reduction in HDAC interaction increases marrow progenitor cell proliferation.
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PMID:Phosphorylation of RUNX1 by cyclin-dependent kinase reduces direct interaction with HDAC1 and HDAC3. 2105 42

Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.
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PMID:MRG15 activates the cdc2 promoter via histone acetylation in human cells. 2132 23

Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.
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PMID:P53-dependent antiproliferative and pro-apoptotic effects of trichostatin A (TSA) in glioblastoma cells. 2227 Aug 49

We have previously reported the identification of a novel WD-domain protein, STRAP that plays a role in maintenance of mesenchymal morphology by regulating E-cadherin and that enhances tumorigenicity partly by downregulating CDK inhibitor p21(Cip1). However, the functional mechanism of regulation of E-cadherin and p21(Cip1) by STRAP is unknown. Here, we have employed STRAP knock out and knockdown cell models (mouse embryonic fibroblast, human cancer cell lines) to show how STRAP downregulates E-cadherin and p21(Cip1) by abrogating the binding of Sp1 to its consensus binding sites. Moreover, ChIP assays suggest that STRAP recruits HDAC1 to Sp1 binding sites in p21(Cip1) promoter. Interestingly, loss of STRAP can stabilize Sp1 by repressing its ubiquitination in G1 phase, resulting in an enhanced expression of p21(Cip1) by >4.5-fold and cell cycle arrest. Using Bioinformatics and Microarray analyses, we have observed that 87% mouse genes downregulated by STRAP have conserved Sp1 binding sites. In NSCLC, the expression levels of STRAP inversely correlated with that of Sp1 (60%). These results suggest a novel mechanism of regulation of E-cadherin and p21(Cip1) by STRAP by modulating Sp1-dependent transcription, and higher expression of STRAP in lung cancer may contribute to downregulation of E-cadherin and p21(Cip1) and to tumor progression.
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PMID:Oncogenic STRAP functions as a novel negative regulator of E-cadherin and p21(Cip1) by modulating the transcription factor Sp1. 2548 64


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