Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Checkpoint kinase 1 (Chk1) mediates diverse cellular responses to genotoxic stress, regulating the network of genome-surveillance pathways that coordinate cell cycle progression with DNA repair. Chk1 is essential for mammalian development and viability, and has been shown to be important for both S and G(2) checkpoints. We now present evidence that the HTLV-1 Tax protein interacts directly with Chk1 and impairs its kinase activities in vitro and in vivo. The direct and physical interaction of Chk1 and Tax was observed in HTLV-1-infected T cells (C81, HuT 102 and MT-2) and transfected fibroblasts (293 T) by coimmunoprecipitation and by in vitro GST pull-down assays. Interestingly, Tax inhibited the kinase activity of Chk1 protein in in vitro and in vivo kinase assays. Consistent with these results, Tax inhibited the phosphorylation-dependent degradation of Cdc25A and G(2) arrest in response to gamma-irradiation (IR) in a dose-dependent manner in vivo. The G(2) arrest did not require Chk2 or p53. These studies provide the first example of a viral transforming protein targeting Chk1 and provide important insights into checkpoint pathway regulation.
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PMID:Human T-cell leukemia virus type 1 Tax interacts with Chk1 and attenuates DNA-damage induced G2 arrest mediated by Chk1. 1510 32

The transcriptional co-activator CBP [CREB (cAMP-response-element-binding protein)-binding protein] and its paralogue p300 play a key role in the regulation of both activity and stability of the tumour suppressor p53. Degradation of p53 is mediated by the ubiquitin ligase MDM2 (mouse double minute protein) and is also reported to be regulated by CBP/p300. Direct protein-protein interaction between a central domain of MDM2 and the TAZ1 (transcriptional adaptor zinc-binding domain) [C/H1 (cysteine/histidine-rich region 1)] domain of p300 and subsequent formation of a ternary complex including p53 have been reported previously. We expressed and purified the proposed binding domains of HDM2 (human homologue of MDM2) and CBP, and examined their interactions using CD spectroscopy. The binding studies were extended by using natively purified GST (glutathione S-transferase)-p300 TAZ1 and GST-p53 fusion proteins, together with in vitro translated HDM2 fragments, under similar solution conditions to those in previous studies, but omitting added EDTA, which causes unfolding and aggregation of the zinc-binding TAZ1 domain. Comparing the binding properties of the known TAZ1 interaction partners HIF-1alpha (hypoxia-inducible factor 1), CITED2 (CBP/p300-interacting transactivator with glutamic- and aspartic-rich tail) and STAT2 (signal transducer and activator of transcription 2) with HDM2, our data suggest that TAZ1 in its native state does not serve as a specific recognition domain of HDM2. Rather, unfolded TAZ1 and HDM2 proteins have a high tendency to aggregate, and non-specific protein complexes are formed under certain conditions.
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PMID:The CBP/p300 TAZ1 domain in its native state is not a binding partner of MDM2. 1527 Jul

We examined the impact of purified bacterially synthesized GST-MDA-7 (IL-24) and ionizing radiation on the proliferation and survival of nonestablished human glioblastoma multiforme (GBM) cells. Glioma cell types expressing mutated PTEN and p53 molecules, activated ERBB1VIII, overexpressing wild type ERBB1 or without receptor overexpression were selected. In MTT assays, GST-MDA-7 caused a dose-dependent reduction in the proliferation of nonestablished glioma cells; however only at higher concentrations did GST-MDA-7 reduce cell viability. The anti-proliferative and cytotoxic effects of GST-MDA-7 were enhanced by radiation in a greater than additive fashion that correlated with JNK1/2/3 activation. The reduction in cell growth and enhancement in cell killing by the combination of GST-MDA-7 and radiation were blocked by an ROS scavenger, N-acetyl cysteine (NAC), a JNK1/2/3 inhibitor SP600125, a pan-caspase inhibitor (zVAD) and by an inhibitor of caspase 9 (LEHD), but not by an inhibitor of caspase 8 (IETD). Low concentrations of either GST-MDA-7 or radiation reduced clonogenic survival, however colony formation ability was significantly further decreased when the two treatments were combined, which was also blocked by inhibition of caspase 9 function. In general agreement with activation of the intrinsic caspase pathway, cell death correlated with reduced BCL-XL expression and with increased levels of the pro-apoptotic proteins BAD and BAX. Inhibition of caspase 9 after combination treatment blunted neither JNK1/2/3 activation nor the enhanced expression of BAD and BAX, but did block caspase 3 cleavage, reduced expression of BCL-XL and inhibition of ERK1/2 activity. In contrast, incubation with NAC blocked JNK1/2/3 activation and cell killing, but not the increases in BAD and BAX expression. These findings argue that after combination treatment JNK1/2/3 activation is a primary pro-apoptotic event and loss of BCL-XL expression and ERK1/2 activity are secondary caspase-dependent processes. This data also argues that GST- MDA-7 induces two parallel pro-apoptotic pathways via ROS-dependent and -independent mechanisms. Infection of primary human astrocytes with a recombinant adenovirus to express MDA-7, Ad.mda-7, but not infection with either Ad.cmv or Ad.mda-7SP- lacking MDA-7 secretion, resulted in the suppression of GBM cell colony formation in soft agar overlay assays, an effect that was enhanced in a greater than additive fashion by radiation. Collectively, our findings demonstrate that MDA-7 reduces proliferation and enhances the radiosensitivity of nonestablished human GBM cells in vitro, and when grown in 3 dimensions, and that sensitization occurs independently of basal EGFR/ERK1/2/AKT activity or the functions of PTEN and p53.
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PMID:MDA-7 regulates cell growth and radiosensitivity in vitro of primary (non-established) human glioma cells. 1532 89

The ataxia-telangiectasia mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date.
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PMID:ATM protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation. 1531 75

Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses. Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER). We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts. GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His)6-tagged p53 proteins, indicating direct protein-protein interaction between those proteins. Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1. These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms.
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PMID:Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen. 1535 33

Upon a certain DNA damage including cisplatin treatment, p73 is stabilized and exerts its growth-suppressive and/or proapoptotic function. However, the precise molecular basis by which the intracellular levels of p73 are regulated remains unclear. In the present study, we have identified RanBPM as a novel binding partner of p73alpha by yeast-based two-hybrid screening, and also found that RanBPM has an ability to stabilize p73alpha. GST pull-down assays and co-immunoprecipitation experiments revealed that RanBPM directly bound to the extreme COOH-terminal region of p73alpha, whereas it failed to interact with p53. Co-expression of RanBPM with p73alpha resulted in the nuclear translocation of RanBPM, and both proteins co-localized in cell nucleus as examined by indirect immunofluorescent staining. It is worth noting that the expression of RanBPM inhibited the ubiquitination of p73alpha, and thereby prolonged its half-life. Subsequent studies demonstrated that the proapoptotic activity of p73alpha was significantly enhanced in the presence of RanBPM. Taken together, our present findings implicate a novel role for RanBPM in the regulation of p73 stability and function.
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PMID:Protein stability and function of p73 are modulated by a physical interaction with RanBPM in mammalian cultured cells. 1555 19

The hepatitis B virus (HBV) protein HBx has been implicated to induce liver cancer in transgenic mice and transactivates a variety of viral and cellular promoters. The 17 kDa protein HBx consists of 154 amino acids, contains 10 cysteine residues and is translated during the viral infection. It has been shown previously that the HBx protein is able to bind to singlestranded DNA and RNA. This nucleic acid binding activity might be relevant for HBx oncogenic character. Furthermore, HBx has been reported to interact with a series of cellular proteins, especially with transcription factors, including the tumor suppressor protein p53. To evaluate the importance of the cysteine residues in HBx for its interaction with RNA and p53 we expressed full-length HBx-wt as well as several truncated mini-HBx(18-142) proteins with multiple cysteine to serine point mutations as 6xHis fusion proteins in Escherichia coli. Using UV cross-linking assays we demonstrate that all truncated mini-HBx proteins with cysteine/serine point mutations maintained the ability to bind to an AU-38 RNA oligonucleotide. Furthermore, we performed in vitro binding assays of selected HBx mutants with GST-p53, circular dichroism spectroscopic analysis of the mutant HBx protein secondary structure and a p53 based transcription activation assay in yeast cells. In summary, our data suggest that the cysteine residues in the HBx protein are of minor importance for its interaction with both RNA and the p53 protein.
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PMID:The cysteine residues of the hepatitis B virus onco-protein HBx are not required for its interaction with RNA or with human p53. 1568 Oct 62

In the present study, we have investigated mechanisms of transcriptional co-operation between proteins that belong to the tumour suppressor p53 and Sp (specificity protein) families of transcription factors. Such mechanisms may play an important role in the regulation of genes containing binding sites for both classes of transcription factors in their promoters. Two of these genes were analysed in the present study: the cyclin-dependent kinase inhibitor p21Cip1 gene and the PUMA (p53-up-regulated mediator of apoptosis) gene. We found that Sp1 and Sp3, but not Sp2, co-operate functionally with p53, p73 and p63 for the synergistic transactivation of the p21Cip1 promoter in Drosophila Schneider SL2 cells that lack endogenous Sp factors. We also found that Sp1 strongly transactivated the PUMA promoter synergistically with p53, whereas deletion of the Sp1-binding sites abolished the transactivation by p53. Using p53 mutant forms in GST (glutathione S-transferase) pull-down assays, we found that the C-terminal 101 amino acids of p53, which include the oligomerization and regulatory domains of the protein, are required for the physical interactions with Sp1 and Sp3, and that deletion of this region abolished transactivation of the p21Cip1 promoter. Utilizing truncated forms of Sp1, we established that p53 interacted with the two transactivation domains A and B, as well as the DNA-binding domain. Our findings suggest that Sp factors are essential for the cellular responses to p53 activation by genotoxic stress. Understanding in detail how members of the p53 and Sp families of transcription factors interact and work together in the p53-mediated cellular responses may open new horizons in cancer chemotherapy.
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PMID:Physical and functional interactions between members of the tumour suppressor p53 and the Sp families of transcription factors: importance for the regulation of genes involved in cell-cycle arrest and apoptosis. 1579 Mar 10

By GST pull downs and co-immunoprecipitation analyses we found that recombinant Chk2 and HDM2 can form stable complexes in vitro. Chk2/HDM2 complexes were also detected in transfected Cos-1 cells over-expressing both proteins. Furthermore, we show that HDM2, as would be expected, severely affects the Chk2-catalyzed phosphorylation of p53. HDM2 itself is only slightly phosphorylated by Chk2. However, whereas HDM2 inhibits the Chk2-catalyzed p53 phosphorylation, HDM2 phosphorylation by Chk2 doubles in the presence of p53. The significance of the HDM2 phosphorylation is unknown, but it is possible that it might influence the stability of the HDM2/p53 complex.
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PMID:HDM2 negatively affects the Chk2-mediated phosphorylation of p53. 1586 97

ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53 and negatively regulate the cell growth with significant G2/M arrest of cell cycle in HepG2 cells through upregulation of p53-inducible gene p21. However, which region of ING4 could have contributed to the binding to p53 remains largely unclear. Herein, the GST-pulldown experiments revealed that the middle region of ING4, a potential bipartite nuclear localization signal (NLS), could be involved in the binding to p53. Furthermore, the interaction of ING4 to p53 was abrogated in vitro and in vivo when certain mutations or the entire deletion of the NLS domain occurred. More interestingly, the mutations of the NLS domain could alter the ING4 nuclear localization, disrupt the interaction of ING4 with p53, and even, deregulate the p53-inducible gene p21 in MCF-7 cells. All data indicated that the NLS domain of ING4 is essential for the binding of ING4 to p53 and the function of ING4 associated with p53.
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PMID:Nuclear localization signal of ING4 plays a key role in its binding to p53. 1588 81


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